Common Light Chain Mouse

ABSTRACT

A genetically modified mouse is provided, wherein the mouse expresses an immunoglobulin light chain repertoire characterized by a limited number of light chain variable domains. Mice are provided that express just one or a few immunoglobulin light chain variable domains from a limited repertoire in their germline. Methods for making light chain variable regions in mice, including human light chain variable regions, are provided. Methods for making human variable regions suitable for use in multispecific binding proteins, e.g., bispecific antibodies, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 13/093,156filed Apr. 25, 2011, which is a continuation-in-part of U.S. Ser. No.13/022,759 filed Feb. 8, 2011, which is a nonprovisional application ofU.S. Provisional Application Ser. No. 61/302,282, filed Feb. 8, 2010;which applications are hereby incorporated by reference in theirentirety.

FIELD

A genetically modified mouse is provided that expresses antibodieshaving a common human variable/mouse constant light chain associatedwith diverse human variable/mouse constant heavy chains. A method formaking a human bispecific antibody from human variable region genesequences of B cells of the mouse is provided.

BACKGROUND

Antibodies typically comprise a homodimeric heavy chain component,wherein each heavy chain monomer is associated with an identical lightchain. Antibodies having a heterodimeric heavy chain component (e.g.,bispecific antibodies) are desirable as therapeutic antibodies. Butmaking bispecific antibodies having a suitable light chain componentthat can satisfactorily associate with each of the heavy chains of abispecific antibody has proved problematic.

In one approach, a light chain might be selected by surveying usagestatistics for all light chain variable domains, identifying the mostfrequently employed light chain in human antibodies, and pairing thatlight chain in vitro with the two heavy chains of differing specificity.

In another approach, a light chain might be selected by observing lightchain sequences in a phage display library (e.g., a phage displaylibrary comprising human light chain variable region sequences, e.g., ahuman scFv library) and selecting the most commonly used light chainvariable region from the library. The light chain can then be tested onthe two different heavy chains of interest.

In another approach, a light chain might be selected by assaying a phagedisplay library of light chain variable sequences using the heavy chainvariable sequences of both heavy chains of interest as probes. A lightchain that associates with both heavy chain variable sequences might beselected as a light chain for the heavy chains.

In another approach, a candidate light chain might be aligned with theheavy chains' cognate light chains, and modifications are made in thelight chain to more closely match sequence characteristics common to thecognate light chains of both heavy chains. If the chances ofimmunogenicity need to be minimized, the modifications preferably resultin sequences that are present in known human light chain sequences, suchthat proteolytic processing is unlikely to generate a T cell epitopebased on parameters and methods known in the art for assessing thelikelihood of immunogenicity (i.e., in silico as well as wet assays).

All of the above approaches rely on in vitro methods that subsume anumber of a priori restraints, e.g., sequence identity, ability toassociate with specific pre-selected heavy chains, etc. There is a needin the art for compositions and methods that do not rely on manipulatingin vitro conditions, but that instead employ more biologically sensibleapproaches to making human epitope-binding proteins that include acommon light chain.

SUMMARY

Genetically modified mice that express human immunoglobulin heavy andlight chain variable domains, wherein the mice have a limited lightchain variable repertoire, are provided. A biological system forgenerating a human light chain variable domain that associates andexpresses with a diverse repertoire of affinity-matured human heavychain variable domains is provided. Methods for making binding proteinscomprising immunoglobulin variable domains are provided, comprisingimmunizing mice that have a limited immunoglobulin light chainrepertoire with an antigen of interest, and employing an immunoglobulinvariable region gene sequence of the mouse in a binding protein thatspecifically binds the antigen of interest. Methods include methods formaking human immunoglobulin heavy chain variable domains suitable foruse in making multi-specific antigen-binding proteins.

Genetically engineered mice are provided that select suitableaffinity-matured human immunoglobulin heavy chain variable domainsderived from a repertoire of unrearranged human heavy chain variableregion gene segments, wherein the affinity-matured human heavy chainvariable domains associate and express with a single human light chainvariable domain derived from one human light chain variable region genesegment. Genetically engineered mice that present a choice of two humanlight chain variable region gene segments are also provided.

Genetically engineered mice are provided that express a limitedrepertoire of human light chain variable domains, or a single humanlight chain variable domain, from a limited repertoire of human lightchain variable region gene segments. The mice are genetically engineeredto include a single unrearranged human light chain variable region genesegment (or two human light chain variable region gene segments) thatrearranges to form a rearranged human light chain variable region gene(or two rearranged light chain variable region genes) that express asingle light chain (or that express either or both of two light chains).The rearranged human light chain variable domains are capable of pairingwith a plurality of affinity-matured human heavy chains selected by themice, wherein the heavy chain variable regions specifically binddifferent epitopes.

Genetically engineered mice are provided that express a limitedrepertoire of human light chain variable domains, or a single humanlight chain variable domain, from a limited repertoire of human lightchain variable region sequences. The mice are genetically engineered toinclude a single V/J human light chain sequence (or two V/J sequences)that express a variable region of a single light chain (or that expresseither or both of two variable regions). A light chain comprising thevariable sequence is capable of pairing with a plurality ofaffinity-matured human heavy chains clonally selected by the mice,wherein the heavy chain variable regions specifically bind differentepitopes.

In one aspect, a genetically modified mouse is provided that comprises asingle human immunoglobulin light chain variable (V_(L)) region genesegment that is capable of rearranging with a human J gene segment(selected from one or a plurality of J_(L) segments) and encoding ahuman V_(L) domain of an immunoglobulin light chain. In another aspect,the mouse comprises no more than two human V_(L) gene segments, each ofwhich is capable of rearranging with a human J gene segment (selectedfrom one or a plurality of J_(L) segments) and encoding a human V_(L)domain of an immunoglobulin light chain.

In one embodiment, the single human V_(L) gene segment is operablylinked to a human J_(L) gene segment selected from Jκ1, Jκ2, Jκ3, Jκ4,and Jκ5, wherein the single human V_(L) gene segment is capable ofrearranging to form a sequence encoding a light chain variable regiongene with any of the one or more human J_(L) gene segments.

In one embodiment, the genetically modified mouse comprises animmunoglobulin light chain locus that does not comprise an endogenousmouse V_(L) gene segment that is capable of rearranging to form animmunoglobulin light chain gene, wherein the V_(L) locus comprises asingle human V_(L) gene segment that is capable of rearranging to encodea V_(L) region of a light chain gene. In a specific embodiment, thehuman V_(L) gene segment is a human Vκ1-39Jκ5 gene segment or a humanVκ3-20Jκ1 gene segment. In one embodiment, the genetically modifiedmouse comprises a V_(L) locus that does not comprise an endogenous mouseV_(L) gene segment that is capable of rearranging to form animmunoglobulin light chain gene, wherein the V_(L) locus comprises nomore than two human V_(L) gene segments that are capable of rearrangingto encode a V_(L) region of a light chain gene. In a specificembodiment, the no more than 2 human V_(L) gene segments are a humanVκ1-39Jκ5 gene segment and a human Vκ3-20Jκ1 gene segment.

In one aspect, a genetically modified mouse is provided that comprises asingle rearranged (V/J) human immunoglobulin light chain variable(V_(L)) region (i.e., a V_(L)/J_(L) region) that encodes a human V_(L)domain of an immunoglobulin light chain. In another aspect, the mousecomprises no more than two rearranged human V_(L) regions that arecapable of encoding a human V_(L) domain of an immunoglobulin lightchain.

In one embodiment, the V_(L) region is a rearranged human Vκ1-39/Jsequence or a rearranged human Vκ3-20/J sequence. In one embodiment, thehuman J_(L) segment of the rearranged V_(L)/J_(L) sequence is selectedfrom Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5. In a specific embodiment, the V_(L)region is a human Vκ1-39Jκ5 sequence or a human Vκ3-20JκK1 sequence. Ina specific embodiment, the mouse has both a human Vκ1-39Jκ5 sequence anda human Vκ3-20Jκ1 sequence.

In one embodiment, the human V_(L) gene segment is operably linked to ahuman or mouse leader sequence. In one embodiment, the leader sequenceis a mouse leader sequence. In a specific embodiment, the mouse leadersequence is a mouse Vκ3-7 leader sequence. In a specific embodiment, theleader sequence is operably linked to an unrearranged human V_(L) genesegment. In a specific embodiment, the leader sequence is operablylinked to a rearranged human V_(L)/J_(L) sequence.

In one embodiment, the V_(L) gene segment is operably linked to animmunoglobulin promoter sequence. In one embodiment, the promotersequence is a human promoter sequence. In a specific embodiment, thehuman immunoglobulin promoter is a human Vκ3-15 promoter. In a specificembodiment, the promoter is operably linked to an unrearranged humanV_(L) gene segment. In a specific embodiment, the promoter is operablylinked to a rearranged human V_(L)/J_(L) sequence.

In one embodiment, the light chain locus comprises a leader sequenceflanked 5′ (with respect to transcriptional direction of a V_(L) genesegment) with a human immunoglobulin promoter and flanked 3′ with ahuman V_(L) gene segment that rearranges with a human J segment andencodes a V_(L) domain of a reverse chimeric light chain comprising anendogenous mouse light chain constant region (C_(L)). In a specificembodiment, the V_(L) gene segment is at the mouse Vκ locus, and themouse C_(L) is a mouse Cκ.

In one embodiment, the light chain locus comprises a leader sequenceflanked 5′ (with respect to transcriptional direction of a V_(L) genesegment) with a human immunoglobulin promoter and flanked 3′ with arearranged human V_(L) region (V_(L)/J_(L) sequence) and encodes a V_(L)domain of a reverse chimeric light chain comprising an endogenous mouselight chain constant region (C_(L)). In a specific embodiment, therearranged human V_(L)/J_(L) sequence is at the mouse kappa (κ) locus,and the mouse C_(L) is a mouse Cκ.

In one embodiment, the V_(L) locus of the modified mouse is a κ lightchain locus, and the κ light chain locus comprises a mouse κ intronicenhancer, a mouse κ3′ enhancer, or both an intronic enhancer and a 3′enhancer.

In one embodiment, the mouse comprises a nonfunctional immunoglobulinlambda (λ) light chain locus. In a specific embodiment, the λ lightchain locus comprises a deletion of one or more sequences of the locus,wherein the one or more deletions renders the λ light chain locusincapable of rearranging to form a light chain gene. In anotherembodiment, all or substantially all of the V_(L) gene segments of the λlight chain locus are deleted.

In one embodiment, mouse makes a light chain that comprises asomatically mutated V_(L) domain derived from a human V_(L) genesegment. In one embodiment, the light chain comprises a somaticallymutated V_(L) domain derived from a human V_(L) gene segment, and amouse Cκ region. In one embodiment, the mouse does not express a λ lightchain.

In one embodiment, the genetically modified mouse is capable ofsomatically hypermutating the human V_(L) region sequence. In a specificembodiment, the mouse comprises a cell that comprises a rearrangedimmunoglobulin light chain gene derived from a human V_(L) gene segmentthat is capable of rearranging and encoding a V_(L) domain, and therearranged immunoglobulin light chain gene comprises a somaticallymutated V_(L) domain.

In one embodiment, the mouse comprises a cell that expresses a lightchain comprising a somatically mutated human V_(L) domain linked to amouse CK, wherein the light chain associates with a heavy chaincomprising a somatically mutated V_(H) domain derived from a human V_(H)gene segment and wherein the heavy chain comprises a mouse heavy chainconstant region (C_(H)). In a specific embodiment, the heavy chaincomprises a mouse C_(H)1, a mouse hinge, a mouse C_(H)2, and a mouseC_(H)3. In a specific embodiment, the heavy chain comprises a humanC_(H)1, a hinge, a mouse C_(H)2, and a mouse C_(H)3.

In one embodiment, the mouse comprises a replacement of endogenous mouseV_(H) gene segments with one or more human V_(H) gene segments, whereinthe human V_(H) gene segments are operably linked to a mouse C_(H)region gene, such that the mouse rearranges the human V_(H) genesegments and expresses a reverse chimeric immunoglobulin heavy chainthat comprises a human V_(H) domain and a mouse C_(H). In oneembodiment, 90-100% of unrearranged mouse V_(H) gene segments arereplaced with at least one unrearranged human V_(H) gene segment. In aspecific embodiment, all or substantially all of the endogenous mouseV_(H) gene segments are replaced with at least one unrearranged humanV_(H) gene segment. In one embodiment, the replacement is with at least19, at least 39, or at least 80 or 81 unrearranged human V_(H) genesegments. In one embodiment, the replacement is with at least 12functional unrearranged human V_(H) gene segments, at least 25functional unrearranged human V_(H) gene segments, or at least 43functional unrearranged human V_(H) gene segments. In one embodiment,the mouse comprises a replacement of all mouse D_(H) and J_(H) segmentswith at least one unrearranged human D_(H) segment and at least oneunrearranged human J_(H) segment. In one embodiment, the at least oneunrearranged human D_(H) segment is selected from 1-1, D1-7, 1-26, 2-8,2-15, 3-3, 3-10, 3-16, 3-22, 5-5, 5-12, 6-6, 6-13, 7-27, and acombination thereof. In one embodiment, the at least one unrearrangedhuman J_(H) segment is selected from 1, 2, 3, 4, 5, 6, and a combinationthereof. In a specific embodiment, the one or more human V_(H) genesegment is selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11,3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51,a 6-1 human V_(H) gene segment, and a combination thereof.

In one embodiment, the mouse comprises a B cell that expresses a bindingprotein that specifically binds an antigen of interest, wherein thebinding protein comprises a light chain derived from a human Vκ1-39/Jκ5rearrangement or a human Vκ3-20/Jκ1 rearrangement, and wherein the cellcomprises a rearranged immunoglobulin heavy chain gene derived from arearrangement of human V_(H) gene segments selected from a 1-69, 2-5,3-13, 3-23, 3-30, 3-33, 3-53, 4-39, 4-59, and 5-51 gene segment. In oneembodiment, the one or more human V_(H) gene segments are rearrangedwith a human heavy chain J_(H) gene segment selected from 1, 2, 3, 4, 5,and 6. In one embodiment, the one or more human V_(H) and J_(H) genesegments are rearranged with a human D_(H) gene segment selected from1-1, 1-7, 1-26, 2-8, 2-15, 3-3, 3-10, 3-16, 3-22, 5-5, 5-12, 6-6, 6-13,and 7-27. In a specific embodiment, the light chain gene has 1, 2, 3, 4,or 5 or more somatic hypermutations.

In one embodiment, the mouse comprises a B cell that comprises arearranged immunoglobulin heavy chain variable region gene sequencecomprising a V_(H)/D_(H)/J_(H) region selected from 2-5/6-6/1,2-5/3-22/1, 3-13/6-6/5, 3-23/2-8/4, 3-23/3-3/4, 3-23/3-10/4, 3-23/6-6/4,3-23/7-27/4, 3-30/1-1/4, 3-30/1-7/4, 3-30/3-3/3, 3-30/3-3/4,3-30/3-22/5, 3-30/5-5/2, 3-30/5-12/4, 3-30/6-6/1, 3-30/6-6/3,3-30/6-6/4, 3-30/6-6/5, 3-30/6-13/4, 3-30/7-27/4, 3-30/7-27/5,3-30/7-27/6, 3-33/1-7/4, 3-33/2-15/4, 4-39/1-26/3, 4-59/3-16/3,4-59/3-16/4, 4-59/3-22/3, 5-51/3-16/6, 5-51/5-5/3, 5-51/6-13/5,3-53/1-1/4, 1-69/6-6/5, and 1-69/6-13/4. In a specific embodiment, the Bcell expresses a binding protein comprising a human immunoglobulin heavychain variable region fused with a mouse heavy chain constant region,and a human immunoglobulin light chain variable region fused with amouse light chain constant region.

In one embodiment, the rearranged human V_(L) region is a humanVκ1-39Jκ5 sequence, and the mouse expresses a reverse chimeric lightchain comprising (i) a V_(L) domain derived from the human V_(L)/J_(L)sequence and (ii) a mouse C_(L); wherein the light chain is associatedwith a reverse chimeric heavy chain comprising (i) a mouse C_(H) and(ii) a somatically mutated human V_(H) domain derived from a human V_(H)gene segment selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11,3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51,a 6-1 human V_(H) gene segment, and a combination thereof. In oneembodiment, the mouse expresses a light chain that is somaticallymutated. In one embodiment the C_(L) is a mouse Cκ. In a specificembodiment, the human V_(H) gene segment is selected from a 2-5, 3-13,3-23, 3-30, 4-59, 5-51, and 1-69 gene segment. In a specific embodiment,the somatically mutated human V_(H) domain comprises a sequence derivedfrom a D_(H) segment selected from 1-1, 1-7, 2-8, 3-3, 3-10, 3-16, 3-22,5-5, 5-12, 6-6, 6-13, and 7-27. In a specific embodiment, thesomatically mutated human V_(H) domain comprises a sequence derived froma J_(H) segment selected from 1, 2, 3, 4, 5, and 6. In a specificembodiment, the somatically mutated human V_(H) domain is encoded by arearranged human V_(H)/D_(H)/J_(H) sequence selected from 2-5/6-6/1,2-5/3-22/1, 3-13/6-6/5, 3-23/2-8/4, 3-23/3-3/4, 3-23/3-10/4, 3-23/6-6/4,3-23/7-27/4, 3-30/1-1/4, 3-30/1-7/4, 3-30/3-3/4, 3-30/3-22/5,3-30/5-5/2, 3-30/5-12/4, 3-30/6-6/1, 3-30/6-6/3, 3-30/6-6/4, 3-30/6-6/5,3-30/6-13/4, 3-30/7-27/4, 3-30/7-27/5, 3-30/7-27/6, 4-59/3-16/3,4-59/3-16/4, 4-59/3-22/3, 5-51/5-5/3, 1-69/6-6/5, and 1-69/6-13/4.

In one embodiment, the rearranged human V_(L) region is a humanVκ3-20Jκ1 sequence, and the mouse expresses a reverse chimeric lightchain comprising (i) a V_(L) domain derived from the rearranged humanV_(L)/J_(L) sequence, and (ii) a mouse C_(L); wherein the light chain isassociated with a reverse chimeric heavy chain comprising (i) a mouseC_(H), and (ii) a somatically mutated human V_(H) derived from a humanV_(H) gene segment selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9,3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59,5-51, a 6-1 human V_(H) gene segment, and a combination thereof. In oneembodiment, the mouse expresses a light chain that is somaticallymutated. In one embodiment the C_(L) is a mouse Cκ. In a specificembodiment, the human V_(H) gene segment is selected from a 3-30, 3-33,3-53, 4-39, and 5-51 gene segment. In a specific embodiment, thesomatically mutated human V_(H) domain comprises a sequence derived froma D_(H) segment selected from 1-1, 1-7, 1-26, 2-15, 3-3, 3-16, and 6-13.In a specific embodiment, the somatically mutated human V_(H) domaincomprises a sequence derived from a J_(H) segment selected from 3, 4, 5,and 6. In a specific embodiment, the somatically mutated human V_(H)domain is encoded by a rearranged human V_(H)/D_(H)/J_(H) sequenceselected from 3-30/1-1/4, 3-30/3-3/3, 3-33/1-7/4, 3-33/2-15/4,4-39/1-26/3, 5-51/3-16/6, 5-51/6-13/5, and 3-53/1-1/4.

In one embodiment, the mouse comprises both a rearranged human Vκ1-39Jκ5sequence and a rearranged human Vκ3-20Jκ1 sequence, and the mouseexpresses a reverse chimeric light chain comprising (i) a V_(L) domainderived from the human Vκ1-39Jκ5 sequence or the human Vκ3-20Jκ1sequence, and (ii) a mouse C_(L); wherein the light chain is associatedwith a reverse chimeric heavy chain comprising (i) a mouse C_(H), and(ii) a somatically mutated human V_(H) derived from a human V_(H) genesegment selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11, 3-13,3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51, a 6-1human V_(H) gene segment, and a combination thereof. In one embodiment,the mouse expresses a light chain that is somatically mutated. In oneembodiment the C_(L) is a mouse Cκ.

In one embodiment, 90-100% of the endogenous unrearranged mouse V_(H)gene segments are replaced with at least one unrearranged human V_(H)gene segment. In a specific embodiment, all or substantially all of theendogenous unrearranged mouse V_(H) gene segments are replaced with atleast one unrearranged human V_(H) gene segment. In one embodiment, thereplacement is with at least 18, at least 39, at least 80, or 81unrearranged human V_(H) gene segments. In one embodiment, thereplacement is with at least 12 functional unrearranged human V_(H) genesegments, at least 25 functional unrearranged human V_(H) gene segments,or at least 43 unrearranged human V_(H) gene segments.

In one embodiment, the genetically modified mouse is a C57BL strain, ina specific embodiment selected from C57BL/A, C57BL/An, C57BL/GrFa,C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In a specific embodiment, thegenetically modified mouse is a mix of an aforementioned 129 strain andan aforementioned C57BL/6 strain. In another specific embodiment, themouse is a mix of aforementioned 129 strains, or a mix of aforementionedBL/6 strains. In a specific embodiment, the 129 strain of the mix is a129S6 (129/SvEvTac) strain.

In one embodiment, the mouse expresses a reverse chimeric antibodycomprising a light chain that comprises a mouse Cκ and a somaticallymutated human V_(L) domain derived from a rearranged human Vκ1-39Jκ5sequence or a rearranged human Vκ3-20Jκ1 sequence, and a heavy chainthat comprises a mouse C_(H) and a somatically mutated human V_(H)domain derived from a human V_(H) gene segment selected from a 1-2, 1-8,1-24, 1-69, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33,3-48, 3-53, 4-31, 4-39, 4-59, 5-51, and a 6-1 human V_(H) gene segment,wherein the mouse does not express a fully mouse antibody and does notexpress a fully human antibody. In one embodiment the mouse comprises aκ light chain locus that comprises a replacement of endogenous mouse κlight chain gene segments with the rearranged human Vκ1-39Jκ5 sequenceor the rearranged human Vκ3-20Jκ1 sequence, and comprises a replacementof all or substantially all endogenous mouse V_(H) gene segments with acomplete or substantially complete repertoire of human V_(H) genesegments.

In one aspect, a population of antigen-specific antibodies derived froma mouse as described herein is provided, wherein the antibodies comprisea light chain gene derived from a human Vκ1-39/Jκ5 rearrangement or ahuman Vκ3-20/Jκ1 rearrangement, and wherein the antibodies comprise arearranged immunoglobulin heavy chain gene derived from a rearrangementof a human V_(H) gene segment selected from a 1-2, 1-3, 1-8, 1-18, 1-24,1-46, 1-58, 1-69, 2-5, 2-26, 2-70, 3-7, 3-9, 3-11, 3-13, 3-15, 3-16,3-20, 3-21, 3-23, 3-30, 3-33, 3-43, 3-48, 3-53, 3-64, 3-72, 3-73, 4-31,4-34, 4-39, 4-59, 5-51, and a 6-1 human V_(H) gene segment. In oneembodiment, the one or more human V_(H) gene segments are rearrangedwith a human heavy chain J_(H) gene segment selected from 1, 2, 3, 4, 5,and 6. In a specific embodiment, the light chain has 1, 2, 3, 4, or 5 ormore somatic hypermutations.

In one embodiment, the light chain has 1, 2, 3, or 4 somatichypermutations. In one embodiment, the light chain gene has 1 or 2mutations. In various embodiments, the light chain gene is capable ofincurring multiple mutations along its sequence.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and the light chain has at least one or no more than foursomatic hypermutations. In one embodiment, the light chain comprises atleast two somatic hypermutations. In one embodiment, the light chaincomprises at least three somatic hypermutations. In one embodiment, thelight chain comprises at least four somatic hypermutations. In aspecific embodiment, the mutations are present in one or more frameworkregions (FWs) of the light chain. In a specific embodiment, themutations are present in one or more complementarity determining regions(CDRs) of the light chain. In a specific embodiment, the mutations arepresent in one or more FWs and/or one or more CDRs of the light chain.In various embodiments, the framework regions are selected fromframework 1 (FW1), framework 2 (FW2), framework 3 (FW3), and/or acombination thereof. In various embodiments, the CDRs are selected fromCDR1, CDR2, CDR3, and/or a combination thereof.

In one embodiment, the heavy chain comprises at least one mutation inone or more FWs or one or more CDRs. In one embodiment, the heavy chaincomprises at least one mutation in one or more FWs and one or more CDRs.In one embodiment, the heavy chain comprises at least two mutations inone or more FWs and one or more CDRs. In one embodiment, the heavy chaincomprises at least three mutations in one or more FWs and one or moreCDRs. In one embodiment, the heavy chain comprises at least fourmutations in one or more FWs and one or more CDRs. In one embodiment,the heavy chain comprises at least five or more than five mutations inone or more FWs and one or more CDRs; in a specific embodiment, theheavy chain comprises at least five or more than five mutations in twoFWs; in a specific embodiment, the heavy chain comprises at least fiveor more than five mutations in one FW and one CDR.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 9% of the Vκ1-39/Jκ5-derived light chains haveat least one mutation present in FW1; in one embodiment, at least 9% ofthe light chains comprise one mutation present in FW1. In oneembodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 25% of the Vκ1-39/Jκ5-derived light chains haveat least one or no more than two mutations present in CDR1; in oneembodiment, at least 19% of the light chains have one mutation presentin CDR1; in one embodiment, at least 5% of the light chains have twomutations present in CDR1.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 20% of the Vκ1-39/Jκ5-derived light chains haveat least one or no more than three mutations present in FW2; in oneembodiment, at least 17% of the light chains have one mutation presentin FW2; in one embodiment, at least 1% of the light chains have twomutations present in FW2; in one embodiment, at least 1% of the lightchains have three mutations present in FW2.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 10% of the Vκ1-39/Jκ5-derived light chains haveat least one or no more than two mutations present in CDR2; in oneembodiment, at least 10% of the light chains have one mutation presentin CDR2; in one embodiment, at least 1% of the light chains have twomutations present in CDR2.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 29% of the Vκ1-39/Jκ5-derived light chains haveat least one or no more than four mutations present in FW3; in oneembodiment, at least 21% of the light chains have one mutation presentin FW3; in one embodiment, at least 5% of the light chains have twomutations present in FW3; in one embodiment, at least 2% of the lightchains have three mutations present in FW3; in one embodiment, at least2% of the light chains have four mutations present in FW3.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 37% of the Vκ1-39/Jκ5-derived light chains haveat least one or no more than four mutations present in CDR3; in oneembodiment, at least 27% of the light chains have one mutation presentin CDR3; in one embodiment, at least 8% of the light chains have twomutations present in CDR3; in one embodiment, at least 1% of the lightchains have three mutations present in CDR3; in one embodiment, at least1% of the light chains have four mutations present in CDR3.

In one embodiment, a population of antigen-specific antibodies derivedfrom a mouse as described herein is provided, wherein the antibodiescomprise a light chain derived from a human Vκ1-39/Jκ5 rearrangement andabout 9% of the Vκ1-39/Jκ5-derived light chains have one or moremutations present in FW1, about 25% of the Vκ1-39/Jκ5-derived lightchains have one or more mutations present in CDR1, about 20% of theVκ1-39/Jκ5-derived light chains have one or more mutations present inFW2, about 10% of the Vκ1-39/Jκ5-derived light chains have one or moremutations present in CDR2, about 29% of the Vκ1-39/Jκ5-derived lightchains have one or more mutations present in FW3, and about 37% of theVκ1-39/Jκ5-derived light chains have one or more mutations present inCDR3.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 35% of the heavy chains have at least onemutation present in FW1; in one embodiment, at least 25% of the heavychains have one mutation present in FW1; in one embodiment, at least 9%of the heavy chains have two mutations present in FW1; in oneembodiment, at least 1% of the heavy chains have three mutations presentin FW1; in one embodiment, at least 1% of the heavy chains have morethan five mutations present in FW1.

In one embodiment, the light chain is derived from a human Vκ1-39Jκ5rearrangement and about 92% of the heavy chains have at least one or nomore than four mutations present in CDR1; in one embodiment, at least92% of the heavy chains have at least one, at least two, at least three,or at least four mutations present in CDR1; in one embodiment, at least26% of the heavy chains have one mutation present in CDR1; in oneembodiment, at least 44% of the heavy chains have two mutations presentin CDR1; in one embodiment, at least 19% of the heavy chains have threemutations present in CDR1; in one embodiment, at least 3% of the heavychains have four mutations present in CDR1.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 66% of the heavy chains have at least one or nomore than three mutations present in FW2; in one embodiment, at least66% of the heavy chains have at least one, at least two, or at leastthree mutations present in FW2; in one embodiment, at least 35% of theheavy chains have one mutation present in FW2; in one embodiment, atleast 23% of the heavy chains have two mutations present in FW2; in oneembodiment, at least 8% of the heavy chains have three mutations presentin FW2.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 70% of the heavy chains have at least one or nomore than four mutations present in CDR2; in one embodiment, at least70% of the heavy chains have at least two, at least three, or at leastfour mutations present in CDR2; in one embodiment, at least 34% have onemutation present in CDR2; in one embodiment, at least 20% of the heavychains have two mutations present in CDR2; in one embodiment, at least12% of the heavy chains have three mutations present in CDR2; in oneembodiment, at least 5% of the heavy chains have four mutations presentin CDR2.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 91% of the heavy chains have at least one or upto five or more mutations present in FW3; in one embodiment, at least91% of the heavy chains have at least two, at least three, at leastfour, or at least five or more mutations present in FW3; in oneembodiment, at least 19% of the heavy chains have one mutation presentin FW3; in one embodiment, at least 33% of the heavy chains have twomutations present in FW3; in one embodiment, at least 22% of the heavychains have three mutations present in FW3; in one embodiment, at least11% of the heavy chains have four mutations present in FW3; in oneembodiment, at least 7% of the heavy chains have five or more mutationspresent in FW3.

In one embodiment, the light chain is derived from a human Vκ1-39/Jκ5rearrangement and about 63% of the heavy chains have at least one or nomore than two mutations present in CDR3; in one embodiment, at least 63%of the heavy chains have at one mutation present in CDR3; in oneembodiment, at least 54% of the heavy chains have one mutation presentin CDR3; in one embodiment, at least 9% of the heavy chains have twomutations present in CDR3.

In one embodiment, a population of antigen-specific antibodies derivedfrom a mouse as described herein is provided, wherein the antibodiescomprise a light chain derived from a human Vκ1-39/Jκ5 rearrangement andat least 35% of the heavy chains have one or more mutations present inFW1, about 92% of the heavy chains have one or more mutations present inCDR1, about 66% of the heavy chains have one or more mutations presentin FW2, about 70% of the heavy chains have one or more mutations presentin CDR2, about 91% of the heavy chains have one or more mutationspresent in FW3, and about 63% of the heavy chains have one or moremutations present in CDR3.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and the light chain gene has at least one or no more thantwo somatic hypermutations; in one embodiment, the light chain gene hasat least two, at least three, at least four or more somatichypermutations. In a specific embodiment, the mutations are present inone or more framework regions of the light chain. In a specificembodiment, the mutations are present in one or more CDR regions of thelight chain. In a specific embodiment, the mutations are present in oneor more framework regions and/or one or more CDR regions of the lightchain. In various embodiments, the framework regions are selected fromframework 1 (FW1), framework 2 (FW2), framework 3 (FW3), and/or acombination thereof.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 10% of the Vκ3-20/Jκ1 -derived light chains haveat least one mutation present in FW1; in one embodiment, at least 10% ofthe light chains have one mutation in FW1.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 53% of the Vκ3-20/Jκ1 -derived light chains haveat least one or no more than two mutations present in CDR1; in oneembodiment, at least 27% of the light chains have one or more mutationsin CDR1; in one embodiment, about 54% of the light chains have one ortwo mutations present in CDR1.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 6% of the Vκ3-20/Jκ1-derived light chains haveat least one or no more than two mutations present in FW2; in oneembodiment, at least 6% of light chains have at least one mutationpresent in FW2; in one embodiment, at least 3% of the light chains haveone mutation present in FW2; in one embodiment, at least 3% of the lightchains have two mutations present in FW2.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and at least about 3% of the Vκ3-20/Jκ1 -derived lightchains have at least one mutation present in CDR2; in one embodiment, atleast 3% of the light chains have one mutation in CDR2.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 17% or more of the Vκ3-20/Jκ1-derived lightchains have at least one or no more than two mutations present in FW3;in one embodiment, at least 20% of the light chain have one mutationpresent in FW3; in one embodiment, at least 17% of the light chains havetwo mutations present in FW3.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and at least 43% of the Vκ3-20/Jκ1-derived light chainshave at least one mutation present in CDR3; in one embodiment, at least43% of the light chains have one mutation in CDR3.

In one embodiment, a population of antigen-specific antibodies derivedfrom a mouse as described herein is provided, wherein the antibodiescomprise a light chain derived from a human Vκ3-20/Jκ1 rearrangement andabout 10% of the Vκ3-20/Jκ1-derived light chains have one or moremutations present in at least, about 53% of the Vκ3-20/Jκ1-derived lightchains have one or more mutations present in CDR1, about 6% of theVκ3-20/Jκ1-derived light chains have one or more mutations present inFW2, about 3% of the Vκ3-20/Jκ1-derived light chains have one or moremutations present in CDR2, about 37% of the Vκ3-20/Jκ1 -derived lightchains have one or more mutations present in FW3, and about 43% of theVκ3-20/Jκ1-derived light chains have one or more mutations present inCDR3.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 43% of the heavy chains have at least one or nomore than two mutations present in FW1; in one embodiment, at least 41%of the heavy chains have at least one mutation present in FW1; in oneembodiment, about 41% of the heavy chains have one mutation present inFW1; in one embodiment, about 2% of the heavy chains have two mutationspresent in FW1.

In one embodiment, the light chain is derived from a human Vκ3-20/κ1rearrangement and about 92% of the heavy chains have at least one or nomore than four mutations present in CDR1; in one embodiment, at least43% of heavy chains have at least one mutation present in CDR1; in oneembodiment, at least 25% of heavy chains have at least two mutationspresent in CDR1; in one embodiment, at least 15% of heavy chains have atleast 3 mutations present in CDR1; in one embodiment, at least 10% ofheavy chains have 4 or more mutations present in CDR1.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 46% of the heavy chains have at least one or nomore than three mutations present in FW2; in one embodiment, at least34% of heavy chains have at least one mutation present in FW2; in oneembodiment, at least 10% of heavy chains have two or more mutationspresent in FW2; in one embodiment, at least 2% of heavy chains havethree or more mutations present in FW2.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 84% of the heavy chains have at least one or upto five or more than five mutations present in CDR2; in one embodiment,at least 39% of the heavy chains have one or more mutations present inCDR2; in one embodiment, at least 18% of the heavy chains have two ormore mutations present in CDR2; in one embodiment, at least 21% of theheavy chains have three or more mutations present in CDR2; in oneembodiment, at least 3% of the heavy chains have four or more mutationspresent in CDR2; in one embodiment, at least 2% of the heavy chains havefive or more mutations present in CDR2.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 92% of the heavy chains have at least one or upto five or more than five mutations present in FW3; in one embodiment,at least 21% of the light chains have at least one mutation present inFW3; in one embodiment, at least 20% of heavy chains have at least twomutations present in FW3; in one embodiment, at least 13% of the heavychains have at least three mutations present in FW3; in one embodiment,at least 20% of the heavy chains have at least four mutations in FW3; inone embodiment, at least 18% of the heavy chains have at least 5mutations in FW3.

In one embodiment, the light chain is derived from a human Vκ3-20/Jκ1rearrangement and about 7% of the heavy chains have at least onemutation present in CDR3; in one embodiment, about 7% of the heavychains have one mutation in CDR3.

In one embodiment, a population of antigen-specific antibodies derivedfrom a mouse as described herein is provided, wherein the antibodiescomprise a light chain derived from a human Vκ3-20/Jκ1 rearrangement andabout 43% of the heavy chains have one or more mutations present in FW1,about 92% of the heavy chains have one or more mutations present inCDR1, about 46% of the heavy chains have one or more mutations presentin FW2, about 84% of the heavy chains have one or more mutations presentin CDR2, about 92% of the heavy chains have one or more mutationspresent in FW3, and about 7% of the heavy chains have one or moremutations present in CDR3.

In one aspect, a mouse that expresses an immunoglobulin light chain froma rearranged immunoglobulin light chain sequence is provided, whereinthe rearranged immunoglobulin light chain sequence is present in thegermline of the mouse, wherein the immunoglobulin light chain comprisesa human variable sequence. In one embodiment, the germline of the mousecomprises a rearranged immunoglobulin light chain sequence that isderived from the same V segment and the same J segment as allnon-surrogate light chain sequences present in every B cell of the mousethat comprises a rearranged light chain sequence.

In one embodiment, the germline of the mouse lacks a functionalunrearranged immunoglobulin light chain V gene segment. In oneembodiment, the germline of the mouse lacks a functional unrearrangedimmunoglobulin light chain J gene segment.

In one embodiment, the germline of the mouse comprises no more than one,no more than two, or no more than three rearranged (V/J) light chainsequences.

In one embodiment, the rearranged V/J sequence comprises a κ light chainsequence. In a specific embodiment, the κ light chain sequence is ahuman κ light chain sequence. In a specific embodiment, the κ lightchain sequence is selected from a human Vκ1-39/J sequence, a humanVκ3-20/J sequence, and a combination thereof. In a specific embodiment,the κ light chain sequence is a human Vκ1-39/Jκ5 sequence. In a specificembodiment, the κ light chain sequence is a human Vκ3-20/Jκ1 sequence.

In one embodiment, the mouse further comprises in its germline asequence selected from a mouse κ intronic enhancer 5′ with respect tothe rearranged immunoglobulin light chain sequence, a mouse κ 3′enhancer, and a combination thereof.

In one embodiment, the mouse comprises an unrearranged human V_(H) genesegment, an unrearranged human D_(H) gene segment, and an unrearrangedhuman J_(H) gene segment, wherein said V_(H), D_(H), and J_(H) genesegments are capable of rearranging to form an immunoglobulin heavychain variable gene sequence operably linked to a heavy chain constantgene sequence. In one embodiment, the mouse comprises a plurality ofhuman V_(H), D_(H), and J_(H) gene segments. In a specific embodiment,the human V_(H), D_(H), and J_(H) gene segments replace endogenous mouseV_(H), D_(H), and J_(H) gene segments at the endogenous mouseimmunoglobulin heavy chain locus. In a specific embodiment, the mousecomprises a replacement of all or substantially all functional mouseV_(H), D_(H), and J_(H) gene segments with all or substantially allfunctional human V_(H), D_(H), and J_(H) gene segments.

In one embodiment, the mouse expresses an immunoglobulin light chainthat comprises a mouse constant sequence. In one embodiment, the mouseexpresses an immunoglobulin light chain that comprises a human constantsequence.

In one embodiment, the mouse expresses an immunoglobulin heavy chainthat comprises a mouse sequence selected from a C_(H)1 sequence, a hingesequence, a C_(H)2 sequence, a C_(H)3 sequence, and a combinationthereof.

In one embodiment, the mouse expresses an immunoglobulin heavy chainthat comprises a human sequence selected from a C_(H)1 sequence, a hingesequence, a C_(H)2 sequence, a C_(H)3 sequence, and a combinationthereof.

In one embodiment, the rearranged immunoglobulin light chain sequence inthe germline of the mouse is at an endogenous mouse immunoglobulin lightchain locus. In a specific embodiment, the rearranged immunoglobulinlight chain sequence in the germline of the mouse replaces all orsubstantially all mouse light chain V and J sequences at the endogenousmouse immunoglobulin light chain locus.

In one aspect, a mouse is provided that comprises a B cell populationcharacterized by each B cell that comprises a non-surrogate light chainsequence comprises a rearranged light chain gene that is derived from asingle human V segment and a single human J segment, wherein the onlylight chain variable sequence in the germline of the mouse is arearranged sequence derived from the single human V segment and thesingle human J segment, and wherein each B ell that comprises therearranged light chain gene further comprises a gene encoding a cognatehuman heavy chain variable domain, and wherein the rearranged lightchain gene comprises at least one, at least two, at least three, or atleast four somatic hypermutations.

In one aspect, a pluripotent, induced pluripotent, or totipotent cellderived from a mouse as described herein is provided. In a specificembodiment, the cell is a mouse embryonic stem (ES) cell.

In one aspect, a tissue derived from a mouse as described herein isprovided. In one embodiment, the tissue is derived from spleen, lymphnode or bone marrow of a mouse as described herein.

In one aspect, a nucleus derived from a mouse as described herein isprovided. In one embodiment, the nucleus is from a diploid cell that isnot a B cell.

In one aspect, a mouse cell is provided that is isolated from a mouse asdescribed herein. In one embodiment, the cell is an ES cell. In oneembodiment, the cell is a lymphocyte. In one embodiment, the lymphocyteis a B cell. In one embodiment, the B cell expresses a chimeric heavychain comprising a variable domain derived from a human gene segment;and a light chain derived from a rearranged human Vκ1-39/J sequence,rearranged human Vκ3-20/J sequence, or a combination thereof; whereinthe heavy chain variable domain is fused to a mouse constant region andthe light chain variable domain is fused to a mouse or a human constantregion.

In one aspect, a hybridoma is provided, wherein the hybridoma is madewith a B cell of a mouse as described herein. In a specific embodiment,the B cell is from a mouse as described herein that has been immunizedwith an immunogen comprising an epitope of interest, and the B cellexpresses a binding protein that binds the epitope of interest, thebinding protein has a somatically mutated human V_(H) domain and a mouseC_(H), and has a human V_(L) domain derived from a rearranged humanVκ1-39Jκ5 or a rearranged human Vκ3-20Jκ1 and a mouse C_(L).

In one aspect, a mouse embryo is provided, wherein the embryo comprisesa donor ES cell that is derived from a mouse as described herein.

In one aspect, a targeting vector is provided, comprising, from 5′ to 3′in transcriptional direction with reference to the sequences of the 5′and 3′ mouse homology arms of the vector, a 5′ mouse homology arm, ahuman or mouse immunoglobulin promoter, a human or mouse leadersequence, and a human V_(L) region selected from a rearranged humanVκ1-39Jκ5 or a rearranged human Vκ3-20Jκ1, and a 3′ mouse homology arm.In one embodiment, the 5′ and 3′ homology arms target the vector to asequence 5′ with respect to an enhancer sequence that is present 5′ andproximal to the mouse Cκ gene. In one embodiment, the promoter is ahuman immunoglobulin variable region gene segment promoter. In aspecific embodiment, the promoter is a human Vκ3-15 promoter. In oneembodiment, the leader sequence is a mouse leader sequence. In aspecific embodiment, the mouse leader sequence is a mouse Vκ3-7 leadersequence.

In one aspect, a targeting vector is provided as described above, but inplace of the 5′ mouse homology arm the human or mouse promoter isflanked 5′ with a site-specific recombinase recognition site (SRRS), andin place of the 3′ mouse homology arm the human V_(L) region is flanked3′ with an SRRS.

In one aspect, a reverse chimeric antibody made by a mouse as describedherein, wherein the reverse chimeric antibody comprises a light chaincomprising a human V_(L) and a mouse C_(L), and a heavy chain comprisinga human V_(H) and a mouse C_(H).

In one aspect, a method for making an antibody is provided, comprisingexpressing in a single cell (a) a first V_(H) gene sequence of animmunized mouse as described herein fused with a human C_(H) genesequence; (b) a V_(L) gene sequence of an immunized mouse as describedherein fused with a human C_(L) gene sequence; and, (c) maintaining thecell under conditions sufficient to express a fully human antibody, andisolating the antibody. In one embodiment, the cell comprises a secondV_(H) gene sequence of a second immunized mouse as described hereinfused with a human C_(H) gene sequence, the first V_(H) gene sequenceencodes a V_(H) domain that recognizes a first epitope, and the secondV_(H) gene sequence encodes a V_(H) domain that recognizes a secondepitope, wherein the first epitope and the second epitope are notidentical.

In one aspect, a method for making an epitope-binding protein isprovided, comprising exposing a mouse as described herein with animmunogen that comprises an epitope of interest, maintaining the mouseunder conditions sufficient for the mouse to generate an immunoglobulinmolecule that specifically binds the epitope of interest, and isolatingthe immunoglobulin molecule that specifically binds the epitope ofinterest; wherein the epitope-binding protein comprises a heavy chainthat comprises a somatically mutated human V_(H) and a mouse C_(H),associated with a light chain comprising a mouse C_(L) and a human V_(L)derived from a rearranged human Vκ1-39Jκ5 or a rearranged humanVκ3-20Jκ1.

In one aspect, a cell that expresses an epitope-binding protein isprovided, wherein the cell comprises: (a) a human nucleotide sequenceencoding a human V_(L) domain that is derived from a rearranged humanVκ1-39Jκ5 or a rearranged human Vκ3-20Jκ1, wherein the human nucleotidesequence is fused (directly or through a linker) to a humanimmunoglobulin light chain constant domain cDNA sequence (e.g., a humanκ constant domain DNA sequence); and, (b) a first human V_(H) nucleotidesequence encoding a human V_(H) domain derived from a first human V_(H)nucleotide sequence, wherein the first human V_(H) nucleotide sequenceis fused (directly or through a linker) to a human immunoglobulin heavychain constant domain cDNA sequence; wherein the epitope-binding proteinrecognizes a first epitope. In one embodiment, the epitope-bindingprotein binds the first epitope with a dissociation constant of lowerthan 10⁻⁶ M, lower than 10⁻⁸ M lower than 10⁻⁹ M, lower than 10⁻¹⁰ M,lower than 10⁻¹¹ M, or lower than 10⁻¹² M.

In one embodiment, the cell comprises a second human nucleotide sequenceencoding a second human V_(H) domain, wherein the second human sequenceis fused (directly or through a linker) to a human immunoglobulin heavychain constant domain cDNA sequence, and wherein the second human V_(H)domain does not specifically recognize the first epitope (e.g., displaysa dissociation constant of, e.g., 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, or higher),and wherein the epitope-binding protein recognizes the first epitope andthe second epitope, and wherein the first and the second immunoglobulinheavy chains each associate with an identical light chain of (a).

In one embodiment, the second V_(H) domain binds the second epitope witha dissociation constant that is lower than 10⁻⁶ M, lower than 10⁻⁷M,lower than 10⁻⁸ M, lower than 10⁻⁹ M, lower than 10⁻¹⁰ M, lower than10⁻¹¹ M, or lower than 10⁻¹² M.

In one embodiment, the epitope-binding protein comprises a firstimmunoglobulin heavy chain and a second immunoglobulin heavy chain, eachassociated with an identical light chain derived from a rearranged humanV_(L) region selected from a human Vκ1-39Jκ5 or a human Vκ3-20Jκ1,wherein the first immunoglobulin heavy chain binds a first epitope witha dissociation constant in the nanomolar to picomolar range, the secondimmunoglobulin heavy chain binds a second epitope with a dissociationconstant in the nanomolar to picomolar range, the first epitope and thesecond epitope are not identical, the first immunoglobulin heavy chaindoes not bind the second epitope or binds the second epitope with adissociation constant weaker than the micromolar range (e.g., themillimolar range), the second immunoglobulin heavy chain does not bindthe first epitope or binds the first epitope with a dissociationconstant weaker than the micromolar range (e.g., the millimolar range),and one or more of the V_(L), the V_(H) of the first immunoglobulinheavy chain, and the V_(H) of the second immunoglobulin heavy chain, aresomatically mutated.

In one embodiment, the first immunoglobulin heavy chain comprises aprotein A-binding residue, and the second immunoglobulin heavy chainlacks the protein A-binding residue.

In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and aretinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™cell).

In one aspect, a reverse chimeric antibody is provided, comprising ahuman V_(H) and a mouse heavy chain constant domain, a human V_(L) and amouse light chain constant domain, wherein the antibody is made by aprocess that comprises immunizing a mouse as described herein with animmunogen comprising an epitope, and the antibody specifically binds theepitope of the immunogen with which the mouse was immunized. In oneembodiment, the V_(L) domain is somatically mutated. In one embodimentthe V_(H) domain is somatically mutated. In one embodiment, both theV_(L) domain and the V_(H) domain are somatically mutated. In oneembodiment, the V_(L) is linked to a mouse Cκ domain.

In one aspect, a mouse is provided, comprising human V_(H) gene segmentsreplacing all or substantially all mouse V_(H) gene segments at theendogenous mouse heavy chain locus; no more than one or two rearrangedhuman light chain V_(L)/J_(L) sequences selected from a rearrangedVκ1-39/J and a rearranged Vκ3-20/J or a combination thereof, replacingall mouse light chain gene segments; wherein the human heavy chainvariable gene segments are linked to a mouse constant gene, and therearranged human light chain sequences are linked to a human or mouseconstant gene.

In one aspect, a mouse ES cell comprising a replacement of all orsubstantially all mouse heavy chain variable gene segments with humanheavy chain variable gene segments, and no more than one or tworearranged human light chain V_(L)/J_(L) sequences, wherein the humanheavy chain variable gene segments are linked to a mouse immunoglobulinheavy chain constant gene, and the rearranged human light chainV_(L)/J_(L) sequences are linked to a mouse or human immunoglobulinlight chain constant gene. In a specific embodiment, the light chainconstant gene is a mouse constant gene.

In one aspect, an antigen-binding protein made by a mouse as describedherein is provided. In a specific embodiment, the antigen-bindingprotein comprises a human immunoglobulin heavy chain variable regionfused with a mouse constant region, and a human immunoglobulin lightchain variable region derived from a Vκ1-39 gene segment or a Vκ3-20gene segment, wherein the light chain constant region is a mouseconstant region.

In one aspect, a fully human antigen-binding protein made from animmunoglobulin variable region gene sequence from a mouse as describedherein is provided, wherein the antigen-binding protein comprises afully human heavy chain comprising a human variable region derived froma sequence of a mouse as described herein, and a fully human light chaincomprising a Vκ1-39 or a Vκ3-20. In one embodiment, the light chainvariable region comprises one to five somatic mutations. In oneembodiment, the light chain variable region is a cognate light chainvariable region that is paired in a B cell of the mouse with the heavychain variable region.

In one embodiment, the fully human antigen-binding protein comprises afirst heavy chain and a second heavy chain, wherein the first heavychain and the second heavy chain comprise non-identical variable regionsindependently derived from a mouse as described herein, and wherein eachof the first and second heavy chains express from a host cell associatedwith a human light chain derived from a Vκ1-39 gene segment or a Vκ3-20gene segment. In one embodiment, the first heavy chain comprises a firstheavy chain variable region that specifically binds a first epitope of afirst antigen, and the second heavy chain comprises a second heavy chainvariable region that specifically binds a second epitope of a secondantigen. In a specific embodiment, the first antigen and the secondantigen are different. In a specific embodiment, the first antigen andthe second antigen are the same, and the first epitope and the secondepitope are not identical; in a specific embodiment, binding of thefirst epitope by a first molecule of the binding protein does not blockbinding of the second epitope by a second molecule of the bindingprotein.

In one aspect, a fully human binding protein derived from a humanimmunoglobulin sequence of a mouse as described herein comprises a firstimmunoglobulin heavy chain and a second immunoglobulin heavy chain,wherein the first immunoglobulin heavy chain comprises a first variableregion that is not identical to a variable region of the secondimmunoglobulin heavy chain, and wherein the first immunoglobulin heavychain comprises a wild type protein A binding determinant, and thesecond heavy chain lacks a wild type protein A binding determinant. Inone embodiment, the first immunoglobulin heavy chain binds protein Aunder isolation conditions, and the second immunoglobulin heavy chaindoes not bind protein A or binds protein A at least 10-fold, ahundred-fold, or a thousand-fold weaker than the first immunoglobulinheavy chain binds protein A under isolation conditions. In a specificembodiment, the first and the second heavy chains are IgG1 isotypes,wherein the second heavy chain comprises a modification selected from95R (EU 435R), 96F (EU 436F), and a combination thereof, and wherein thefirst heavy chain lacks such modification.

In one aspect, a method for making a bispecific antigen-binding proteinis provided, comprising exposing a first mouse as described herein to afirst antigen of interest that comprises a first epitope, exposing asecond mouse as described herein to a second antigen of interest thatcomprises a second epitope, allowing the first and the second mouse toeach mount immune responses to the antigens of interest, identifying inthe first mouse a first human heavy chain variable region that binds thefirst epitope of the first antigen of interest, identifying in thesecond mouse a second human heavy chain variable region that binds thesecond epitope of the second antigen of interest, making a first fullyhuman heavy chain gene that encodes a first heavy chain that binds thefirst epitope of the first antigen of interest, making a second fullyhuman heavy chain gene that encodes a second heavy chain that binds thesecond epitope of the second antigen of interest, expressing the firstheavy chain and the second heavy chain in a cell that expresses a singlefully human light chain derived from a human Vκ1-39 or a human Vκ3-20gene segment to form a bispecific antigen-binding protein, and isolatingthe bispecific antigen-binding protein.

In one embodiment, the first antigen and the second antigen are notidentical.

In one embodiment, the first antigen and the second antigen areidentical, and the first epitope and the second epitope are notidentical. In one embodiment, binding of the first heavy chain variableregion to the first epitope does not block binding of the second heavychain variable region to the second epitope.

In one embodiment, the first antigen is selected from a soluble antigenand a cell surface antigen (e.g., a tumor antigen), and the secondantigen comprises a cell surface receptor. In a specific embodiment, thecell surface receptor is an immunoglobulin receptor. In a specificembodiment, the immunoglobulin receptor is an Fc receptor. In oneembodiment, the first antigen and the second antigen are the same cellsurface receptor, and binding of the first heavy chain to the firstepitope does not block binding of the second heavy chain to the secondepitope.

In one embodiment, the light chain variable domain of the light chaincomprises 2 to 5 somatic mutations. In one embodiment, the light chainvariable domain is a somatically mutated cognate light chain expressedin a B cell of the first or the second immunized mouse with either thefirst or the second heavy chain variable domain.

In one embodiment, the first fully human heavy chain bears an amino acidmodification that reduces its affinity to protein A, and he second fullyhuman heavy chain does not comprise a modification that reduces itsaffinity to protein A.

In one aspect, an antibody or a bispecific antibody comprising a humanheavy chain variable domain made in accordance with the invention isprovided. In another aspect, use of a mouse as described herein to makea fully human antibody or a fully human bispecific antibody is provided.

In one aspect, a genetically modified mouse, embryo, or cell describedherein comprises a κ light chain locus that retains endogenousregulatory or control elements, e.g., a mouse κ intronic enhancer, amouse κ 3′ enhancer, or both an intronic enhancer and a 3′ enhancer,wherein the regulatory or control elements facilitate somatic mutationand affinity maturation of an expressed sequence of the κ light chainlocus.

In one aspect, a mouse is provided that comprises a B cell populationcharacterized by having immunoglobulin light chains derived from no morethan one, or no more than two, rearranged or unrearranged immunoglobulinlight chain V and J gene segments, wherein the mouse exhibits a κ:λlight chain ratio that is about the same as a mouse that comprises awild type complement of immunoglobulin light chain V and J genesegments.

In one embodiment, the immunoglobulin light chains are derived from nomore than one, or no more than two, rearranged immunoglobulin lightchain V and J gene segments. In a specific embodiment, the light chainsare derived from no more than one rearranged immunoglobulin light chainV and J gene segments.

In one aspect, a mouse as described herein is provided that expresses animmunoglobulin light chain derived from no more than one, or no morethan two, human Vκ/Jκ sequences, wherein the mouse comprises areplacement of all or substantially all endogenous mouse heavy chainvariable region gene segments with one or more human heavy chainvariable region gene segments, and the mouse exhibits a ratio of (a)CD19⁺ B cells that express an immunoglobulin having a λ light chain, to(b) CD19⁺ B cells that express an immunoglobulin having a κ light chain,of about 1 to about 20.

In one embodiment, the mouse expresses a single κ light chain derivedfrom a human Vκ1-39Jκ5 sequence, and the ratio of CD19⁺ B cells thatexpress an immunoglobulin having a 2 light chain to CD19⁺ B cells thatexpress an immunoglobulin having a κ light chain is about 1 to about 20;in one embodiment, the ratio is about 1 to at least about 66; in aspecific embodiment, the ratio is about 1 to 66.

In one embodiment, the mouse expresses a single κ light chain derivedfrom a human Vκ3-20Jκ5 sequence, and the ratio of CD19⁺ B cells thatexpress an immunoglobulin having a λ light chain to CD19⁺ B cells thatexpress an immunoglobulin having a κ light chain is about 1 to about 20;in one embodiment, the ratio is about 1 to about 21. In specificembodiments, the ratio is 1 to 20, or 1 to 21.

In one aspect, a genetically modified mouse is provided that expresses asingle rearranged κ light chain, wherein the mouse comprises afunctional λ light chain locus, and wherein the mouse expresses a B cellpopulation that comprises Igκ⁺ cells that express a κ light chainderived from the same single rearranged κ light chain. In oneembodiment, the percent of Igκ⁺Igλ⁺ B cells in the mouse is about thesame as in a wild type mouse. In a specific embodiment, the percent ofIgκ⁺Igλ⁺ B cells in the mouse is about 2 to about 6 percent. In aspecific embodiment, the percent of Igκ⁺Igλ⁺ B cells in a mouse whereinthe single rearranged κ light chain is derived from a Vκ1-39Jκ5 sequenceis about 2 to about 3; in a specific embodiment, the percent is about2.6. In a specific embodiment, the percent of Igκ⁺Igλ⁺ B cells in amouse wherein the single rearranged κ light chain is derived from aVκ3-20Jκ1 sequence is about 4 to about 8; in a specific embodiment,about 6.

In one aspect, a genetically modified mouse is provided, wherein themouse expresses a single rearranged κ light chain derived from a humanVκ and Jκ gene segment, wherein the mouse expresses a B cell populationthat comprises a single κ light chain derived from the single rearrangedκ light chain sequence, wherein the genetically modified mouse has notbeen rendered resistant to somatic hypermutations. In one embodiment, atleast 90% of the κ light chains expressed on a B cell of the mouseexhibit from at least one to about five somatic hypermutations.

In one aspect, a genetically modified mouse is provided that is modifiedto express a single κ light chain derived from no more than one, or nomore than two, rearranged κ light chain sequences, wherein the mouseexhibits a κ light chain usage that is about two-fold or more, at leastabout three-fold or more, or at least about four-fold or more greaterthan the κ light chain usage exhibited by a wild type mouse, or greaterthan the κ light chain usage exhibited by a mouse of the same strainthat comprises a wild type repertoire of κ light chain gene segments. Ina specific embodiment, the mouse expresses the single κ light chain fromno more than one rearranged κ light chain sequence. In a more specificembodiment, the rearranged κ light chain sequence is selected from aVκ1-39Jκ5 and Vκ3-20Jκ1 sequence. In one embodiment, the rearranged κlight chain sequence is a Vκ1-39Jκ5 sequence. In one embodiment, therearranged κ light chain sequence is a Vκ3-20Jκ1 sequence.

In one aspect, a genetically modified mouse is provided that expresses asingle κ light chain derived from no more than one, or no more than two,rearranged κ light chain sequences, wherein the mouse exhibits a κ lightchain usage that is about 100-fold or more, at least about 200-fold ormore, at least about 300-fold or more, at least about 400-fold or more,at least about 500-fold or more, at least about 600-fold or more, atleast about 700-fold or more, at least about 800-fold or more, at leastabout 900-fold or more, at least about 1000-fold or more greater thanthe same κ light chain usage exhibited by a mouse bearing a complete orsubstantially complete human κ light chain locus. In a specificembodiment, the mouse bearing a complete or substantially complete humanκ light chain locus lacks a functional unrearranged mouse κ light chainsequence. In a specific embodiment, the mouse expresses the single κlight chain from no more than one rearranged κ light chain sequence. Inone embodiment, the mouse comprises one copy of a rearranged κ lightchain sequence (e.g., a heterozygote). In one embodiment, the mousecomprises two copies of a rearranged κ light chain sequence (e.g., ahomozygote). In a more specific embodiment, the rearranged κ light chainsequence is selected from a Vκ1-39Jκ5 and Vκ3-20Jκ1 sequence. In oneembodiment, the rearranged K light chain sequence is a Vκ1-39Jκ5sequence. In one embodiment, the rearranged κ light chain sequence is aVκ3-20Jκ1 sequence.

In one aspect, a genetically modified mouse is provided that expresses asingle light chain derived from no more than one, or no more than two,rearranged light chain sequences, wherein the light chain in thegenetically modified mouse exhibits a level of expression that is atleast 10-fold to about 1,000-fold, 100-fold to about 1,000-fold,200-fold to about 1,000-fold, 300-fold to about 1,000-fold, 400-fold toabout 1,000-fold, 500-fold to about 1,000-fold, 600-fold to about1,000-fold, 700-fold to about 1,000-fold, 800-fold to about 1,000-fold,or 900-fold to about 1,000-fold higher than expression of the samerearranged light chain exhibited by a mouse bearing a complete orsubstantially complete light chain locus. In one embodiment, the lightchain comprises a human sequence. In a specific embodiment, the humansequence is a κ sequence. In one embodiment, the human sequence is a λsequence. In one embodiment, the light chain is a fully human lightchain.

In one embodiment, the level of expression is characterized byquantitating mRNA of transcribed light chain sequence, and comparing itto transcribed light chain sequence of a mouse bearing a complete orsubstantially complete light chain locus.

In one aspect, a genetically modified mouse is provided that expresses asingle κ light chain derived from no more than one, or no more than two,rearranged κ light chain sequences, wherein the mouse, upon immunizationwith antigen, exhibits a serum titer that is comparable to a wild typemouse immunized with the same antigen. In a specific embodiment, themouse expresses a single κ light chain from no more than one rearrangedκ light chain sequence. In one embodiment, the serum titer ischaracterized as total immunoglobulin. In a specific embodiment, theserum titer is characterized as IgM specific titer. In a specificembodiment, the serum titer is characterized as IgG specific titer. In amore specific embodiment, the rearranged κ light chain sequence isselected from a Vκ1-39Jκ5 and Vκ3-20Jκ1 sequence. In one embodiment, therearranged κ light chain sequence is a Vκ1-39Jκ5 sequence. In oneembodiment, the rearranged κ light chain sequence is a Vκ3-20Jκ1sequence.

In one aspect, a genetically modified mouse is provided that expresses apopulation of antigen-specific antibodies, wherein all of theimmunoglobulin light chains of the population of antigen-specificantibodies comprise a human light chain variable (V_(L)) region derivedfrom the same single human V_(L) gene segment and the immunoglobulinheavy chains comprise a human heavy chain variable (V_(H)) regionderived from one of a plurality of human V_(H) gene segments.

In various embodiments, the human V_(H) gene segments are selected fromV_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18, V_(H)1-24, V_(H)1-46,V_(H)1-58, V_(H)1-69, V_(H)2-5, V_(H)2-26, V_(H)2-70, V_(H)3-7,V_(H)3-9, V_(H)3-11, V_(H)3-13, V_(H)3-15, V_(H)3-20, V_(H)3-21,V_(H)3-23, V_(H)3-30, V_(H)3-33, V_(H)3-43, V_(H)3-48, V_(H)3-53,V_(H)3-64, V_(H)3-72, V_(H)3-73, V_(H)4-31, V_(H)4-34, V_(H)4-39,V_(H)4-59, V_(H)5-51, and V_(H)6-1.

In various embodiments, same single human V_(L) gene segment is selectedfrom a human Vκ1-39 gene segment and a human Vκ3-20 gene segment. Invarious embodiments, all of the immunoglobulin light chains comprise ahuman light chain J (J_(L)) gene segment selected from a Jκ and a Jλgene segment. In a specific embodiment, the human J_(L) gene segment isselected from a human Jκ1 and a Jκ5 gene segment. In variousembodiments, the mouse lacks a sequence selected from a mouseimmunoglobulin V_(L) gene segment, a mouse immunoglobulin J_(L) genesegment, and a combination thereof. In various embodiments, the humanV_(L) region is operably linked to a human, mouse, or rat immunoglobulinlight chain constant (C_(L)) region. In a specific embodiment, the humanV_(L) region is operably linked to a mouse Cκ region. In a specificembodiment, the human V_(L) region is operably linked to a rat Cκregion.

In various embodiments, the human V_(L) region is expressed from anendogenous immunoglobulin light chain locus. In various embodiments, thehuman V_(H) region is operably linked to a human, mouse, or ratimmunoglobulin heavy chain constant (C_(H)) region. In variousembodiments the (C_(H)) region comprises a human sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, a C_(H)4, and/or a combinationthereof. In various embodiments, the human V_(H) region is expressedfrom an endogenous immunoglobulin heavy chain locus.

In one aspect, a genetically modified mouse is provided that expresses aplurality of immunoglobulin heavy chains associated with a single lightchain. In one embodiment, the heavy chain comprises a human sequence. Invarious embodiments, the human sequence is selected from a variablesequence, a C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combinationthereof. In one embodiment, the single light chain comprises a humansequence. In various embodiments, the human sequence is selected from avariable sequence, a constant sequence, and a combination thereof. Inone embodiment, the mouse comprises a disabled endogenous immunoglobulinlocus and expresses the heavy chain and/or the light chain from atransgene or extrachromosomal episome. In one embodiment, the mousecomprises a replacement at an endogenous mouse locus of some or allendogenous mouse heavy chain gene segments (i.e., V, D, J), and/or someor all endogenous mouse heavy chain constant sequences (e.g., C_(H)1,hinge, C_(H)2, C_(H)3, or a combination thereof), and/or some or allendogenous mouse light chain sequences (e.g., V, J, constant, or acombination thereof), with one or more human immunoglobulin sequences.

In one aspect, a mouse suitable for making antibodies that have the samelight chain is provided, wherein all or substantially all antibodiesmade in the mouse are expressed with the same light chain. In oneembodiment, the light chain is expressed from an endogenous light chainlocus.

In one aspect, a method for making a light chain for a human antibody isprovided, comprising obtaining from a mouse as described herein a lightchain sequence and a heavy chain sequence, and employing the light chainsequence and the heavy chain sequence in making a human antibody. In oneembodiment, the human antibody is a bispecific antibody.

In one aspect, a method for identifying a human heavy chain variabledomain that is capable of binding an antigen of interest with anengineered light chain as described herein is provided, wherein themethod comprises providing a heavy chain variable domain derived from afirst antibody that is capable of binding the antigen, repairing theheavy chain variable domain with a germline light chain sequence andtransfecting a cell so that each are expressed to form a secondantibody, exposing the second antibody to the antigen, and measuringbinding of the second antibody to the antigen.

In one embodiment, the light chain of the first antibody comprises ahuman Vκ1-39 sequence. In one embodiment, the light chain of the firstantibody comprises a human Vκ3-20 sequence. In one embodiment, thegermline light chain sequence comprises a human Vκ1-39 or Vκ3-20sequence. In various embodiments, binding of the second antibody to theantigen is determined by comparison of binding of the first antibody tothe antigen.

Any of the embodiments and aspects described herein can be used inconjunction with one another, unless otherwise indicated or apparentfrom the context. Other embodiments will become apparent to thoseskilled in the art from a review of the ensuing description.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 illustrates a targeting strategy for replacing endogenous mouseimmunoglobulin light chain variable region gene segments with a humanVκ1-39Jκ5 gene region.

FIG. 2 illustrates a targeting strategy for replacing endogenous mouseimmunoglobulin light chain variable region gene segments with a humanVκ3-20Jκ1 gene region.

FIG. 3 illustrates a targeting strategy for replacing endogenous mouseimmunoglobulin light chain variable region gene segments with a humanVpreB/Jλ5 gene region.

FIG. 4 shows the percent of CD19⁺ B cells (y-axis) from peripheral bloodfor wild type mice (WT), mice homozygous for an engineered humanrearranged Vκ1-39Jκ5 light chain region (Vκ1-39Jκ5 HO) and micehomozygous for an engineered human rearranged Vκ3-20Jκ1 light chainregion (Vκ3-20Jκ1 HO).

FIG. 5A shows the relative mRNA expression (y-axis) of a Vκ1-39-derivedlight chain in a quantitative PCR assay using probes specific for thejunction of an engineered human rearranged Vκ1-39Jκ5 light chain region(Vκ1-39Jκ5 Junction Probe) and the human Vκ1-39 gene segment (Vκ1-39Probe) in a mouse homozygous for a replacement of the endogenous Vκ andJκ gene segments with human Vκ and Jκ gene segments (Hκ), a wild typemouse (WT), and a mouse heterozygous for an engineered human rearrangedVκ1-39Jκ5 light chain region (Vκ1-39Jκ5 HET). Signals are normalized toexpression of mouse CK. N.D.: not detected.

FIG. 5B shows the relative mRNA expression (y-axis) of a Vκ1-39-derivedlight chain in a quantitative PCR assay using probes specific for thejunction of an engineered human rearranged Vκ1-39Jκ5 light chain region(Vκ1-39Jκ5 Junction Probe) and the human Vκ1-39 gene segment (Vκ1-39Probe) in a mouse homozygous for a replacement of the endogenous Vκ andJκ gene segments with human Vκ and Jκ gene segments (Hκ), a wild typemouse (WT), and a mouse homozygous for an engineered human rearrangedVκ1-39Jκ5 light chain region (Vκ1-39Jκ5 HO). Signals are normalized toexpression of mouse Cκ.

FIG. 5C shows the relative mRNA expression (y-axis) of a Vκ3-20-derivedlight chain in a quantitative PCR assay using probes specific for thejunction of an engineered human rearranged Vκ3-20Jκ1 light chain region(Vκ3-20Jκ1 Junction Probe) and the human Vκ3-20 gene segment (Vκ3-20Probe) in a mouse homozygous for a replacement of the endogenous Vκ andJκ gene segments with human Vκ and Jκ gene segments (Hκ), a wild typemouse (WT), and a mouse heterozygous (HET) and homozygous (HO) for anengineered human rearranged Vκ3-20Jκ1 light chain region. Signals arenormalized to expression of mouse CK.

FIG. 6A shows IgM (left) and IgG (right) titer in wild type (WT; N=2)and mice homozygous for an engineered human rearranged Vκ1-39Jκ5 lightchain region (Vκ1-39Jκ5 HO; N=2) immunized with β-galatosidase.

FIG. 6B shows total immunoglobulin (IgM, IgG, IgA) titer in wild type(WT; N=5) and mice homozygous for an engineered human rearrangedVκ3-20Jκ1 light chain region (Vκ3-20Jκ1 HO; N=5) immunized withβ-galatosidase.

DETAILED DESCRIPTION

This invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention is defined bythe claims.

Unless defined otherwise, all terms and phrases used herein include themeanings that the terms and phrases have attained in the art, unless thecontrary is clearly indicated or clearly apparent from the context inwhich the term or phrase is used. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned are herebyincorporated by reference in their entirety.

The term “antibody”, as used herein, includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chaincomprises a heavy chain variable (V_(H)) region and a heavy chainconstant region (C_(H)). The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable (V_(L)) region and a light chain constant region (C_(L)).The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) comprises three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may beabbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may beabbreviated as LCDR1, LCDR2 and LCDR3. The term “high affinity” antibodyrefers to an antibody that has a K_(D) with respect to its targetepitope about of 10⁻⁹ M or lower (e.g., about 1×10⁻⁹ M, 1×10⁻¹⁰ M,1×10⁻¹¹ M, or about 1×10⁻¹² M). In one embodiment, K_(D) is measured bysurface plasmon resonance, e.g., BIACORE™; in another embodiment, K_(D)is measured by ELISA.

The phrase “bispecific antibody” includes an antibody capable ofselectively binding two or more epitopes. Bispecific antibodiesgenerally comprise two nonidentical heavy chains, with each heavy chainspecifically binding a different epitope—either on two differentmolecules (e.g., different epitopes on two different immunogens) or onthe same molecule (e.g., different epitopes on the same immunogen). If abispecific antibody is capable of selectively binding two differentepitopes (a first epitope and a second epitope), the affinity of thefirst heavy chain for the first epitope will generally be at least oneto two or three or four or more orders of magnitude lower than theaffinity of the first heavy chain for the second epitope, and viceversa. Epitopes specifically bound by the bispecific antibody can be onthe same or a different target (e.g., on the same or a differentprotein). Bispecific antibodies can be made, for example, by combiningheavy chains that recognize different epitopes of the same immunogen.For example, nucleic acid sequences encoding heavy chain variablesequences that recognize different epitopes of the same immunogen can befused to nucleic acid sequences encoding the same or different heavychain constant regions, and such sequences can be expressed in a cellthat expresses an immunoglobulin light chain. A typical bispecificantibody has two heavy chains each having three heavy chain CDRs,followed by (N-terminal to C-terminal) a C_(H)1 domain, a hinge, aC_(H)2 domain, and a C_(H)3 domain, and an immunoglobulin light chainthat either does not confer epitope-binding specificity but that canassociate with each heavy chain, or that can associate with each heavychain and that can bind one or more of the epitopes bound by the heavychain epitope-binding regions, or that can associate with each heavychain and enable binding or one or both of the heavy chains to one orboth epitopes.

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In some embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In some embodiments,the cell is eukaryotic and is selected from the following cells: CHO(e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell,Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK),HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21),Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell,SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myelomacell, tumor cell, and a cell line derived from an aforementioned cell.In some embodiments, the cell comprises one or more viral genes, e.g., aretinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

The phrase “complementarity determining region,” or the term “CDR,”includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin genes that normally (i.e., in a wild typeanimal) appears between two framework regions in a variable region of alight or a heavy chain of an immunoglobulin molecule (e.g., an antibodyor a T cell receptor). A CDR can be encoded by, for example, a germlinesequence or a rearranged or unrearranged sequence, and, for example, bya naive or a mature B cell or a T cell. A CDR can be somatically mutated(e.g., vary from a sequence encoded in an animal's germline), humanized,and/or modified with amino acid substitutions, additions, or deletions.In some circumstances (e.g., for a CDR3), CDRs can be encoded by two ormore sequences (e.g., germline sequences) that are not contiguous (e.g.,in an unrearranged nucleic acid sequence) but are contiguous in a B cellnucleic acid sequence, e.g., as the result of splicing or connecting thesequences (e.g., V-D-J recombination to form a heavy chain CDR3).

The term “conservative,” when used to describe a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). In general, a conservativeamino acid substitution will not substantially change the functionalproperties of interest of a protein, for example, the ability of avariable region to specifically bind a target epitope with a desiredaffinity. Examples of groups of amino acids that have side chains withsimilar chemical properties include aliphatic side chains such asglycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxylside chains such as serine and threonine; amide-containing side chainssuch as asparagine and glutamine; aromatic side chains such asphenylalanine, tyrosine, and tryptophan; basic side chains such aslysine, arginine, and histidine; acidic side chains such as asparticacid and glutamic acid; and, sulfur-containing side chains such ascysteine and methionine. Conservative amino acids substitution groupsinclude, for example, valine/leucine/isoleucine, phenylalanine/tyrosine,lysine/arginine, alanine/valine, glutamate/aspartate, andasparagine/glutamine. In some embodiments, a conservative amino acidsubstitution can be substitution of any native residue in a protein withalanine, as used in, for example, alanine scanning mutagenesis. In someembodiments, a conservative substitution is made that has a positivevalue in the PAM250 log-likelihood matrix disclosed in Gonnet et al.(1992) Exhaustive Matching of the Entire Protein Sequence Database,Science 256:1443-45, hereby incorporated by reference. In someembodiments, the substitution is a moderately conservative substitutionwherein the substitution has a nonnegative value in the PAM250log-likelihood matrix.

In some embodiments, residue positions in an immunoglobulin light chainor heavy chain differ by one or more conservative amino acidsubstitutions. In some embodiments, residue positions in animmunoglobulin light chain or functional fragment thereof (e.g., afragment that allows expression and secretion from, e.g., a B cell) arenot identical to a light chain whose amino acid sequence is listedherein, but differs by one or more conservative amino acidsubstitutions.

The phrase “epitope-binding protein” includes a protein having at leastone CDR and that is capable of selectively recognizing an epitope, e.g.,is capable of binding an epitope with a K_(D) that is at about onemicromolar or lower (e.g., a K_(D) that is about 1×10⁻⁶ M, 1×10⁻⁷ M,1×10⁻⁹ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, or about 1×10⁻¹² M).Therapeutic epitope-binding proteins (e.g., therapeutic antibodies)frequently require a K_(D) that is in the nanomolar or the picomolarrange.

The phrase “functional fragment” includes fragments of epitope-bindingproteins that can be expressed, secreted, and specifically bind to anepitope with a K_(D) in the micromolar, nanomolar, or picomolar range.Specific recognition includes having a K_(D) that is at least in themicromolar range, the nanomolar range, or the picomolar range.

The term “germline” includes reference to an immunoglobulin nucleic acidsequence in a non-somatically mutated cell, e.g., a non-somaticallymutated B cell or pre-B cell or hematopoietic cell.

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes animmunoglobulin heavy chain constant region sequence from any organism.Heavy chain variable domains include three heavy chain CDRs and four FRregions, unless otherwise specified. Fragments of heavy chains includeCDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has,following the variable domain (from N-terminal to C-terminal), a C_(H)1domain, a hinge, a C_(H)2 domain, and a C_(H)3 domain. A functionalfragment of a heavy chain includes a fragment that is capable ofspecifically recognizing an epitope (e.g., recognizing the epitope witha K_(D) in the micromolar, nanomolar, or picomolar range), that iscapable of expressing and secreting from a cell, and that comprises atleast one CDR.

The term “identity” when used in connection with sequence includesidentity as determined by a number of different algorithms known in theart that can be used to measure nucleotide and/or amino acid sequenceidentity. In some embodiments described herein, identities aredetermined using a ClustalW v. 1.83 (slow) alignment employing an opengap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnetsimilarity matrix (MACVECTOR™ 10.0.2, MacVector Inc., 2008). The lengthof the sequences compared with respect to identity of sequences willdepend upon the particular sequences, but in the case of a light chainconstant domain, the length should contain sequence of sufficient lengthto fold into a light chain constant domain that is capable ofself-association to form a canonical light chain constant domain, e.g.,capable of forming two beta sheets comprising beta strands and capableof interacting with at least one C_(H)1 domain of a human or a mouse. Inthe case of a C_(H)1 domain, the length of sequence should containsequence of sufficient length to fold into a C_(H)1 domain that iscapable of forming two beta sheets comprising beta strands and capableof interacting with at least one light chain constant domain of a mouseor a human.

The phrase “immunoglobulin molecule” includes two immunoglobulin heavychains and two immunoglobulin light chains. The heavy chains may beidentical or different, and the light chains may be identical ordifferent.

The phrase “light chain” includes an immunoglobulin light chain sequencefrom any organism, and unless otherwise specified includes human κ andλ. light chains and a VpreB, as well as surrogate light chains. Lightchain variable (V_(L)) domains typically include three light chain CDRsand four framework (FR) regions, unless otherwise specified. Generally,a full-length light chain includes, from amino terminus to carboxylterminus, a V_(L) domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4,and a light chain constant domain. Light chains include those, e.g.,that do not selectively bind either a first or a second epitopeselectively bound by the epitope-binding protein in which they appear.Light chains also include those that bind and recognize, or assist theheavy chain with binding and recognizing, one or more epitopesselectively bound by the epitope-binding protein in which they appear.Common light chains are those derived from a rearranged human Vκ1-39Jκ5sequence or a rearranged human Vκ3-20Jκ1 sequence, and includesomatically mutated (e.g., affinity matured) versions.

The phrase “micromolar range” is intended to mean 1-999 micromolar; thephrase “nanomolar range” is intended to mean 1-999 nanomolar; the phrase“picomolar range” is intended to mean 1-999 picomolar.

The phrase “somatically mutated” includes reference to a nucleic acidsequence from a B cell that has undergone class-switching, wherein thenucleic acid sequence of an immunoglobulin variable region (e.g., aheavy chain variable domain or including a heavy chain CDR or FRsequence) in the class-switched B cell is not identical to the nucleicacid sequence in the B cell prior to class-switching, such as, forexample, a difference in a CDR or framework nucleic acid sequencebetween a B cell that has not undergone class-switching and a B cellthat has undergone class-switching. “Somatically mutated” includesreference to nucleic acid sequences from affinity-matured B cells thatare not identical to corresponding immunoglobulin variable regionsequences in B cells that are not affinity-matured (i.e., sequences inthe genome of germline cells). The phrase “somatically mutated” alsoincludes reference to an immunoglobulin variable region nucleic acidsequence from a B cell after exposure of the B cell to an epitope ofinterest, wherein the nucleic acid sequence differs from thecorresponding nucleic acid sequence prior to exposure of the B cell tothe epitope of interest. The phrase “somatically mutated” refers tosequences from antibodies that have been generated in an animal, e.g., amouse having human immunoglobulin variable region nucleic acidsequences, in response to an immunogen challenge, and that result fromthe selection processes inherently operative in such an animal.

The term “unrearranged,” with reference to a nucleic acid sequence,includes nucleic acid sequences that exist in the germline of an animalcell.

The phrase “variable domain” includes an amino acid sequence of animmunoglobulin light or heavy chain (modified as desired) that comprisesthe following amino acid regions, in sequence from N-terminal toC-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

Common Light Chain

Prior efforts to make useful multispecific epitope-binding proteins,e.g., bispecific antibodies, have been hindered by variety of problemsthat frequently share a common paradigm: in vitro selection ormanipulation of sequences to rationally engineer, or to engineer throughtrial-and-error, a suitable format for pairing a heterodimericbispecific human immunoglobulin. Unfortunately, most if not all of thein vitro engineering approaches provide largely ad hoc fixes that aresuitable, if at all, for individual molecules. On the other hand, invivo methods for employing complex organisms to select appropriatepairings that are capable of leading to human therapeutics have not beenrealized.

Generally, native mouse sequences are frequently not a good source forhuman therapeutic sequences. For at least that reason, generating mouseheavy chain immunoglobulin variable regions that pair with a commonhuman light chain is of limited practical utility. More in vitroengineering efforts would be expended in a trial-and-error process totry to humanize the mouse heavy chain variable sequences while hoping toretain epitope specificity and affinity while maintaining the ability tocouple with the common human light chain, with uncertain outcome. At theend of such a process, the final product may maintain some of thespecificity and affinity, and associate with the common light chain, butultimately immunogenicity in a human would likely remain a profoundrisk.

Therefore, a suitable mouse for making human therapeutics would includea suitably large repertoire of human heavy chain variable region genesegments in place of endogenous mouse heavy chain variable region genesegments. The human heavy chain variable region gene segments should beable to rearrange and recombine with an endogenous mouse heavy chainconstant domain to form a reverse chimeric heavy chain (i.e., a heavychain comprising a human variable domain and a mouse constant region).The heavy chain should be capable of class switching and somatichypermutation so that a suitably large repertoire of heavy chainvariable domains are available for the mouse to select one that canassociate with the limited repertoire of human light chain variableregions.

A mouse that selects a common light chain for a plurality of heavychains has a practical utility. In various embodiments, antibodies thatexpress in a mouse that can only express a common light chain will haveheavy chains that can associate and express with an identical orsubstantially identical light chain. This is particularly useful inmaking bispecific antibodies. For example, such a mouse can be immunizedwith a first immunogen to generate a B cell that expresses an antibodythat specifically binds a first epitope. The mouse (or a mousegenetically the same) can be immunized with a second immunogen togenerate a B cell that expresses an antibody that specifically binds thesecond epitope. Variable heavy regions can be cloned from the B cellsand expresses with the same heavy chain constant region, and the samelight chain, and expressed in a cell to make a bispecific antibody,wherein the light chain component of the bispecific antibody has beenselected by a mouse to associate and express with the light chaincomponent.

The inventors have engineered a mouse for generating immunoglobulinlight chains that will suitably pair with a rather diverse family ofheavy chains, including heavy chains whose variable regions depart fromgermline sequences, e.g., affinity matured or somatically mutatedvariable regions. In various embodiments, the mouse is devised to pairhuman light chain variable domains with human heavy chain variabledomains that comprise somatic mutations, thus enabling a route to highaffinity binding proteins suitable for use as human therapeutics.

The genetically engineered mouse, through the long and complex processof antibody selection within an organism, makes biologically appropriatechoices in pairing a diverse collection of human heavy chain variabledomains with a limited number of human light chain options. In order toachieve this, the mouse is engineered to present a limited number ofhuman light chain variable domain options in conjunction with a widediversity of human heavy chain variable domain options. Upon challengewith an immunogen, the mouse maximizes the number of solutions in itsrepertoire to develop an antibody to the immunogen, limited largely orsolely by the number or light chain options in its repertoire. Invarious embodiments, this includes allowing the mouse to achievesuitable and compatible somatic mutations of the light chain variabledomain that will nonetheless be compatible with a relatively largevariety of human heavy chain variable domains, including in particularsomatically mutated human heavy chain variable domains.

To achieve a limited repertoire of light chain options, the mouse isengineered to render nonfunctional or substantially nonfunctional itsability to make, or rearrange, a native mouse light chain variabledomain. This can be achieved, e.g., by deleting the mouse's light chainvariable region gene segments. The endogenous mouse locus can then bemodified by an exogenous suitable human light chain variable region genesegment of choice, operably linked to the endogenous mouse light chainconstant domain, in a manner such that the exogenous human variableregion gene segments can combine with the endogenous mouse light chainconstant region gene and form a rearranged reverse chimeric light chaingene (human variable, mouse constant). In various embodiments, the lightchain variable region is capable of being somatically mutated. Invarious embodiments, to maximize ability of the light chain variableregion to acquire somatic mutations, the appropriate enhancer(s) isretained in the mouse. For example, in modifying a mouse κ light chainlocus to replace endogenous mouse κ light chain gene segments with humanκ light chain gene segments, the mouse κ intronic enhancer and mouse κ3′ enhancer are functionally maintained, or undisrupted.

A genetically engineered mouse is provided that expresses a limitedrepertoire of reverse chimeric (human variable, mouse constant) lightchains associated with a diversity of reverse chimeric (human variable,mouse constant) heavy chains. In various embodiments, the endogenousmouse κ light chain gene segments are deleted and replaced with a single(or two) rearranged human light chain region, operably linked to theendogenous mouse Cκ gene. In embodiments for maximizing somatichypermutation of the rearranged human light chain region, the mouse κintronic enhancer and the mouse κ 3′ enhancer are maintained. In variousembodiments, the mouse also comprises a nonfunctional λ light chainlocus, or a deletion thereof or a deletion that renders the locus unableto make a λ light chain.

A genetically engineered mouse is provided that, in various embodiments,comprises a light chain variable region locus lacking endogenous mouselight chain V_(L) and J_(L) gene segments and comprising a rearrangedhuman light chain variable region, in one embodiment a rearranged humanV_(L)/J_(L) sequence, operably linked to a mouse constant region,wherein the locus is capable of undergoing somatic hypermutation, andwherein the locus expresses a light chain comprising the humanV_(L)/J_(L) sequence linked to a mouse constant region. Thus, in variousembodiments, the locus comprises a mouse κ 3′ enhancer, which iscorrelated with a normal, or wild type, level of somatic hypermutation.

The genetically engineered mouse in various embodiments when immunizedwith an antigen of interest generates B cells that exhibit a diversityof rearrangements of human immunoglobulin heavy chain variable regionsthat express and function with one or with two rearranged light chains,including embodiments where the one or two light chains comprise humanlight chain variable regions that comprise, e.g., 1 to 5 somaticmutations. In various embodiments, the human light chains so expressedare capable of associating and expressing with any human immunoglobulinheavy chain variable region expressed in the mouse.

Epitope-binding Proteins Binding More Than One Epitope

The compositions and methods of described herein can be used to makebinding proteins that bind more than one epitope with high affinity,e.g., bispecific antibodies. Advantages of the invention include theability to select suitably high binding (e.g., affinity matured) heavychain immunoglobulin chains each of which will associate with a singlelight chain.

Synthesis and expression of bispecific binding proteins has beenproblematic, in part due to issues associated with identifying asuitable light chain that can associate and express with two differentheavy chains, and in part due to isolation issues. The methods andcompositions described herein allow for a genetically modified mouse toselect, through otherwise natural processes, a suitable light chain thatcan associate and express with more than one heavy chain, includingheavy chains that are somatically mutated (e.g., affinity matured).Human V_(L) and V_(H) sequences from suitable B cells of immunized miceas described herein that express affinity matured antibodies havingreverse chimeric heavy chains (i.e., human variable and mouse constant)can be identified and cloned in frame in an expression vector with asuitable human constant region gene sequence (e.g., a human IgG1). Twosuch constructs can be prepared, wherein each construct encodes a humanheavy chain variable domain that binds a different epitope. One of thehuman V_(L)s (e.g., human Vκ1-39Jκ5 or human Vκ3-20Jκ1), in germlinesequence or from a B cell wherein the sequence has been somaticallymutated, can be fused in frame to a suitable human constant region gene(e.g., a human κ constant gene). These three fully human heavy and lightconstructs can be placed in a suitable cell for expression. The cellwill express two major species: a homodimeric heavy chain with theidentical light chain, and a heterodimeric heavy chain with theidentical light chain. To allow for a facile separation of these majorspecies, one of the heavy chains is modified to omit a Protein A-bindingdeterminant, resulting in a differential affinity of a homodimericbinding protein from a heterodimeric binding protein. Compositions andmethods that address this issue are described in U.S. Ser. No.12/832,838, filed 25 Jun. 2010, entitled “Readily Isolated BispecificAntibodies with Native Immunoglobulin Format,” published as US2010/0331527A1, hereby incorporated by reference.

In one aspect, an epitope-binding protein as described herein isprovided, wherein human V_(L) and V_(H) sequences are derived from micedescribed herein that have been immunized with an antigen comprising anepitope of interest.

In one embodiment, an epitope-binding protein is provided that comprisesa first and a second polypeptide, the first polypeptide comprising, fromN-terminal to C-terminal, a first epitope-binding region thatselectively binds a first epitope, followed by a constant region thatcomprises a first C_(H)3 region of a human IgG selected from IgG1, IgG2,IgG4, and a combination thereof; and, a second polypeptide comprising,from N-terminal to C-terminal, a second epitope-binding region thatselectively binds a second epitope, followed by a constant region thatcomprises a second C_(H)3 region of a human IgG selected from IgG1,IgG2, IgG4, and a combination thereof, wherein the second C_(H)3 regioncomprises a modification that reduces or eliminates binding of thesecond C_(H)3 domain to protein A.

In one embodiment, the second C_(H)3 region comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In anotherembodiment, the second C_(H)3 region further comprises a Y96Fmodification (IMGT; Y436F by EU).

In one embodiment, the second C_(H)3 region is from a modified humanIgG1, and further comprises a modification selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second C_(H)3 region is from a modified humanIgG2, and further comprises a modification selected from the groupconsisting of N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I byEU).

In one embodiment, the second C_(H)3 region is from a modified humanIgG4, and further comprises a modification selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT; Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

One method for making an epitope-binding protein that binds more thanone epitope is to immunize a first mouse in accordance with theinvention with an antigen that comprises a first epitope of interest,wherein the mouse comprises an endogenous immunoglobulin light chainvariable region locus that does not contain an endogenous mouse V_(L)that is capable of rearranging and forming a light chain, wherein at theendogenous mouse immunoglobulin light chain variable region locus is asingle rearranged human V_(L) region operably linked to the mouseendogenous light chain constant region gene, and the rearranged humanV_(L) region is selected from a human Vκ1-39Jκ5 and a human Vκ3-20Jκ1,and the endogenous mouse V_(H) gene segments have been replaced in wholeor in part with human V_(H) gene segments, such that immunoglobulinheavy chains made by the mouse are solely or substantially heavy chainsthat comprise human variable domains and mouse constant domains. Whenimmunized, such a mouse will make a reverse chimeric antibody,comprising only one of two human light chain variable domains (e.g., oneof human Vκ1-39Jκ5 or human Vκ3-20Jκ1). Once a B cell is identified thatencodes a V_(H) that binds the epitope of interest, the nucleotidesequence of the V_(H) (and, optionally, the V_(L)) can be retrieved(e.g., by PCR) and cloned into an expression construct in frame with asuitable human immunoglobulin constant domain. This process can berepeated to identify a second V_(H) domain that binds a second epitope,and a second V_(H) gene sequence can be retrieved and cloned into anexpression vector in frame to a second suitable immunoglobulin constantdomain. The first and the second immunoglobulin constant domains can thesame or different isotype, and one of the immunoglobulin constantdomains (but not the other) can be modified as described herein or in US2010/0331527A1, and epitope-binding protein can be expressed in asuitable cell and isolated based on its differential affinity forProtein A as compared to a homodimeric epitope-binding protein, e.g., asdescribed in US 2010/0331527A1.

In one embodiment, a method for making a bispecific epitope-bindingprotein is provided, comprising identifying a first affinity-matured(e.g., comprising one or more somatic hypermutations) human V_(H)nucleotide sequence (V_(H)1) from a mouse as described herein,identifying a second affinity-matured (e.g., comprising one or moresomatic hypermutations) human V_(H) nucleotide sequence (V_(H)2) from amouse as described herein, cloning V_(H)1 in frame with a human heavychain lacking a Protein A-determinant modification as described in US2010/0331527A1 for form heavy chain 1 (HC1), cloning V_(H)2 in framewith a human heavy chain comprising a Protein A-determinant as describedin US 2010/0331527A1 to form heavy chain 2 (HC2), introducing anexpression vector comprising HC1 and the same or a different expressionvector comprising HC2 into a cell, wherein the cell also expresses ahuman immunoglobulin light chain that comprises a human Vκ1-39/human Jκ5or a human Vκ3-20/human Jκ1 fused to a human light chain constantdomain, allowing the cell to express a bispecific epitope-bindingprotein comprising a V_(H) domain encoded by V_(H)1 and a V_(H) domainencoded by V_(H)2, and isolating the bispecific epitope-binding proteinbased on its differential ability to bind Protein A as compared with amonospecific homodimeric epitope-binding protein. In a specificembodiment, HC1 is an IgG1, and HC2 is an IgG1 that comprises themodification H95R (IMGT; H435R by EU) and further comprises themodification Y96F (IMGT; Y436F by EU). In one embodiment, the VH domainencoded by V_(H)1, the V_(H) domain encoded by V_(H)2, or both, aresomatically mutated.

Human V_(H) Genes That Express with a Common Human V_(L)

A variety of human variable regions from affinity-matured antibodiesraised against four different antigens were expressed with either theircognate light chain, or at least one of a human light chain selectedfrom human Vκ1-39Jκ5, human Vκ3-20Jκ1, or human VpreBJλ5 (see Example1). For antibodies to each of the antigens, somatically mutated highaffinity heavy chains from different gene families paired successfullywith rearranged human germline Vκ1-39Jκ5 and Vκ3-20Jκ1 regions and weresecreted from cells expressing the heavy and light chains. For Vκ1-39Jκ5and Vκ3-20Jκ1, V_(H) domains derived from the following human V_(H) genefamilies expressed favorably: 1-2, 1-8, 1-24, 2-5, 3-7, 3-9, 3-11, 3-13,3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 4-31, 4-39, 4-59, 5-51, and 6-1.Thus, a mouse that is engineered to express a limited repertoire ofhuman V_(L) domains from one or both of Vκ1-39Jκ5 and Vκ3-20Jκ1 willgenerate a diverse population of somatically mutated human V_(H) domainsfrom a V_(H) locus modified to replace mouse V_(H) gene segments withhuman V_(H) gene segments.

Mice genetically engineered to express reverse chimeric (human variable,mouse constant) immunoglobulin heavy chains associated with a singlerearranged light chain (e.g., a Vκ1-39/J or a Vκ3-20/J), when immunizedwith an antigen of interest, generated B cells that comprised adiversity of human V_(H) rearrangements and expressed a diversity ofhigh-affinity antigen-specific antibodies with diverse properties withrespect to their ability to block binding of the antigen to its ligand,and with respect to their ability to bind variants of the antigen (seeExamples 5 through 10).

Thus, the mice and methods described herein are useful in making andselecting human immunoglobulin heavy chain variable domains, includingsomatically mutated human heavy chain variable domains, that result froma diversity of rearrangements, that exhibit a wide variety of affinities(including exhibiting a K_(D) of about a nanomolar or less), a widevariety of specificities (including binding to different epitopes of thesame antigen), and that associate and express with the same orsubstantially the same human immunoglobulin light chain variable region.

The following examples are provided so as to describe to those ofordinary skill in the art how to make and use methods and compositionsof the invention, and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature, etc.)but some experimental errors and deviations should be accounted for.Unless indicated otherwise, parts are parts by weight, molecular weightis average molecular weight, temperature is indicated in Celsius, andpressure is at or near atmospheric.

EXAMPLES

The following examples are provided so as to describe to those ofordinary skill in the art how to make and use methods and compositionsof the invention, and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature, etc.)but some experimental errors and deviations should be accounted for.Unless indicated otherwise, temperature is indicated in Celsius,pressure is at or near atmospheric, parts are by parts by weight, andmolecular weight is average molecular weight.

Example 1 Identification of Human Heavy Chain Variable Regions ThatAssociate With Selected Human Light Chain Variable Regions

An in vitro expression system was constructed to determine if a singlerearranged human germline light chain could be co-expressed with humanheavy chains from antigen specific human antibodies.

Methods for generating human antibodies in genetically modified mice areknown (see e.g., U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals,VELOCIMMUNE®). The VELOCIMMUNE® technology involves generation of agenetically modified mouse having a genome comprising human heavy andlight chain variable regions operably linked to endogenous mouseconstant region loci such that the mouse produces an antibody comprisinga human variable region and a mouse constant region in response toantigenic stimulation. The DNA encoding the variable regions of theheavy and light chains of the antibodies produced from a VELOCIMMUNE®mouse are fully human. Initially, high affinity chimeric antibodies areisolated having a human variable region and a mouse constant region. Asdescribed below, the antibodies are characterized and selected fordesirable characteristics, including affinity, selectivity, epitope,etc. The mouse constant regions are replaced with a desired humanconstant region to generate a fully human antibody containing a non-IgMisotype, for example, wild type or modified IgG1, IgG2, IgG3 or IgG4.While the constant region selected may vary according to specific use,high affinity antigen-binding and target specificity characteristicsreside in the variable region.

A VELOCIMMUNE® mouse was immunized with a growth factor that promotesangiogenesis (Antigen C) and antigen-specific human antibodies wereisolated and sequenced for V gene usage using standard techniquesrecognized in the art. Selected antibodies were cloned onto human heavyand light chain constant regions and 69 heavy chains were selected forpairing with one of three human light chains: (1) the cognate κ lightchain linked to a human κ constant region, (2) a rearranged humangermline Vκ1-39Jκ5 linked to a human κ constant region, or (3) arearranged human germline Vκ3-20Jκ1 linked to a human κ constant region.Each heavy chain and light chain pair were co-transfected in CHO-K1cells using standard techniques. Presence of antibody in the supernatantwas detected by anti-human IgG in an ELISA assay. Antibody titer (ng/ml)was determined for each heavy chain/light chain pair and titers with thedifferent rearranged germline light chains were compared to the titersobtained with the parental antibody molecule (i.e., heavy chain pairedwith cognate light chain) and percent of native titer was calculated(Table 1). V_(H): Heavy chain variable gene. ND: no expression detectedunder current experimental conditions.

TABLE 1 Antibody Titer (ng/mL) Percent of Native Titer Cognate Vκ3-V_(H) LC Vκ1-39Jκ5 Vκ3-20Jκ1 Vκ1-39Jκ5 20Jκ1 3-15 63 23 11 36.2 17.5 1-2103 53 ND 51.1 — 3-23 83 60 23 72.0 27.5 3-33 15 77 ND 499.4 — 4-31 2269 17 309.4 76.7 3-7 53 35 28 65.2 53.1 — 22 32 19 148.8 89.3 1-24 3 13ND 455.2 — 3-33 1 47 ND 5266.7 — 3-33 58 37 ND 63.1 — — 110 67 18 60.616.5 3-23 127 123 21 96.5 16.3 3-33 28 16 2 57.7 7.1 3-23 32 50 38 157.1119.4 — 18 45 18 254.3 101.7 3-9 1 30 23 2508.3 1900.0 3-11 12 26 6225.9 48.3 1-8 16 ND 13 — 81.8 3-33 54 81 10 150.7 19.1 — 34 9 ND 25.9 —3-20 7 14 54 203.0 809.0 3-33 19 38 ND 200.5 — 3-11 48 ND 203 — 423.6 —11 23 8 212.7 74.5 3-33 168 138 182 82.0 108.2 3-20 117 67 100 57.5 86.13-23 86 61 132 70.7 154.1 3-33 20 12 33 60.9 165.3 4-31 69 92 52 133.875.0 3-23 87 78 62 89.5 71.2 1-2 31 82 51 263.0 164.6 3-23 53 93 151175.4 285.4 — 11 8 17 75.7 151.4 3-33 114 36 27 31.6 23.4 3-15 73 39 4453.7 59.6 3-33 1 34 16 5600.0 2683.3 3-9 58 112 57 192.9 97.6 3-33 67 20105 30.1 157.0 3-33 34 21 24 62.7 70.4 3-20 10 49 91 478.4 888.2 3-33 6632 25 48.6 38.2 3-23 17 59 56 342.7 329.8 — 58 108 19 184.4 32.9 — 68 5420 79.4 29.9 3-33 42 35 32 83.3 75.4 — 29 19 13 67.1 43.9 3-9 24 34 29137.3 118.4 3-30/33 17 33 7 195.2 43.1 3-7 25 70 74 284.6 301.6 3-33 87127 ND 145.1 — 6-1 28 56 ND 201.8 — 3-33 56 39 20 69.9 36.1 3-33 10 53 1520.6 6.9 3-33 20 67 10 337.2 52.3 3-33 11 36 18 316.8 158.4 3-23 12 4232 356.8 272.9 3-33 66 95 15 143.6 22.5 3-15 55 68 ND 123.1 — — 32 68 3210.9 10.6 1-8 28 48 ND 170.9 — 3-33 124 192 21 154.3 17.0 3-33 0 113 ND56550.0 — 3-33 10 157 1 1505.8 12.5 3-33 6 86 15 1385.5 243.5 3-23 70115 22 163.5 31.0 3-7 71 117 21 164.6 29.6 3-33 82 100 47 122.7 57.1 3-7124 161 41 130.0 33.5

In a similar experiment, VELOCIMMUNE® mice were immunized with severaldifferent antigens and selected heavy chains of antigen specific humanantibodies were tested for their ability to pair with differentrearranged human germline light chains (as described above). Theantigens used in this experiment included an enzyme involved incholesterol homeostasis (Antigen A), a serum hormone involved inregulating glucose homeostasis (Antigen B), a growth factor thatpromotes angiogenesis (Antigen C) and a cell-surface receptor (AntigenD). Antigen specific antibodies were isolated from mice of eachimmunization group and the heavy chain and light chain variable regionswere cloned and sequenced. From the sequence of the heavy and lightchains, V gene usage was determined and selected heavy chains werepaired with either their cognate light chain or a rearranged humangermline Vκ1-39Jκ5 region. Each heavy/light chain pair wasco-transfected in CHO-K1 cells and the presence of antibody in thesupernatant was detected by anti-human IgG in an ELISA assay. Antibodytiter (μg/ml) was determined for each heavy chain/light chain pairingand titers with the different rearranged human germline light chainswere compared to the titers obtained with the parental antibody molecule(i.e., heavy chain paired with cognate light chain) and percent ofnative titer was calculated (Table 2). V_(H): Heavy chain variable gene.Vκ: κ light chain variable gene. ND: no expression detected undercurrent experimental conditions.

TABLE 2 Titer (μg/ml) Percent of Anti- V_(H) V_(H) + V_(H) + NativeAntigen body V_(H) Vκ Alone Vκ Vκ1-39Jκ5 Titer A 320 1-18 2-30 0.3 3.12.0 66 321 2-5  2-28 0.4 0.4 1.9 448 334 2-5  2-28 0.4 2.7 2.0 73 3133-13 3-15 0.5 0.7 4.5 670 316 3-23 4-1  0.3 0.2 4.1 2174 315 3-30 4-1 0.3 0.2 3.2 1327 318 4-59 1-17 0.3 4.6 4.0 86 B 257 3-13 1-5  0.4 3.13.2 104 283 3-13 1-5  0.4 5.4 3.7 69 637 3-13 1-5  0.4 4.3 3.0 70 6383-13 1-5  0.4 4.1 3.3 82 624 3-23 1-17 0.3 5.0 3.9 79 284 3-30 1-17 0.34.6 3.4 75 653 3-33 1-17 0.3 4.3 0.3 7 268 4-34 1-27 0.3 5.5 3.8 69 6334-34 1-27 0.6 6.9 3.0 44 C 730 3-7  1-5  0.3 1.1 2.8 249 728 3-7  1-5 0.3 2.0 3.2 157 691 3-9  3-20 0.3 2.8 3.1 109 749 3-33 3-15 0.3 3.8 2.362 750 3-33 1-16 0.3 3.0 2.8 92 724 3-33 1-17 0.3 2.3 3.4 151 706 3-331-16 0.3 3.6 3.0 84 744 1-18 1-12 0.4 5.1 3.0 59 696 3-11 1-16 0.4 3.02.9 97 685 3-13 3-20 0.3 0.5 3.4 734 732 3-15 1-17 0.3 4.5 3.2 72 6943-15 1-5  0.4 5.2 2.9 55 743 3-23 1-12 0.3 3.2 0.3 10 742 3-23 2-28 0.44.2 3.1 74 693 3-23 1-12 0.5 4.2 4.0 94 D 136 3-23 2-28 0.4 5.0 2.7 55155 3-30 1-16 0.4 1.0 2.2 221 163 3-30 1-16 0.3 0.6 3.0 506 171 3-301-16 0.3 1.0 2.8 295 145 3-43 1-5  0.4 4.4 2.9 65 49 3-48 3-11 0.3 1.72.6 155 51 3-48 1-39 0.1 1.9 0.1 4 159 3-7  6-21 0.4 3.9 3.6 92 169 3-7 6-21 0.3 1.3 3.1 235 134 3-9  1-5  0.4 5.0 2.9 58 141 4-31 1-33 2.4 4.22.6 63 142 4-31 1-33 0.4 4.2 2.8 67

The results obtained from these experiments demonstrate that somaticallymutated, high affinity heavy chains from different gene families areable to pair with rearranged human germline Vκ1-39κ5 and Vκ3-20Jκ1regions and be secreted from the cell as a normal antibody molecule. Asshown in Table 1, antibody titer was increased for about 61% (42 of 69)heavy chains when paired with the rearranged human Vκ1-39Jκ5 light chainand about 29% (20 of 69) heavy chains when paired with the rearrangedhuman Vκ3-20Jκ1 light chain as compared to the cognate light chain ofthe parental antibody. For about 20% (14 of 69) of the heavy chains,both rearranged human germline light chains conferred an increase inexpression as compared to the cognate light chain of the parentalantibody. As shown in Table 2, the rearranged human germline Vκ1-39Jκ5region conferred an increase in expression of several heavy chainsspecific for a range of different classes of antigens as compared to thecognate light chain for the parental antibodies. Antibody titer wasincreased by more than two-fold for about 35% (15/43) of the heavychains as compared to the cognate light chain of the parentalantibodies. For two heavy chains (315 and 316), the increase was greaterthan ten-fold as compared to the parental antibody. Within all the heavychains that showed increase expression relative to the cognate lightchain of the parental antibody, family three (V_(H)3) heavy chains areover represented in comparison to other heavy chain variable region genefamilies. This demonstrates a favorable relationship of human V_(H)3heavy chains to pair with rearranged human germline Vκ1-39Jκ5 andVκ3-20Jκ1 light chains.

Example 2 Generation of a Rearranged Human Germline Light Chain Locus

Various rearranged human germline light chain targeting vectors weremade using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251and Valenzuela et al. (2003) High-throughput engineering of the mousegenome coupled with high-resolution expression analysis, Nature Biotech.21(6):652-659) to modify mouse genomic Bacterial Artificial Chromosome(BAC) clones 302g12 and 254m04 (Invitrogen). Using these two BAC clones,genomic constructs were engineered to contain a single rearranged humangermline light chain region and inserted into an endogenous κ lightchain locus that was previously modified to delete the endogenous κvariable and joining gene segments.

Construction of Rearranged Human Germline Light Chain Targeting Vectors.Three different rearranged human germline light chain regions were madeusing standard molecular biology techniques recognized in the art. Thehuman variable gene segments used for constructing these three regionsincluded rearranged human Vκ1-39Jκ5 sequence, a rearranged humanVκ3-20Jκ1 sequence and a rearranged human VpreBJλ5 sequence.

A DNA segment containing exon 1 (encoding the leader peptide) and intron1 of the mouse Vκ3-7 gene was made by de novo DNA synthesis (IntegratedDNA Technologies). Part of the 5′ untranslated region up to a naturallyoccurring Blpl restriction enzyme site was included. Exons of humanVκ1-39 and Vκ3-20 genes were PCR amplified from human genomic BAClibraries. The forward primers had a 5′ extension containing the spliceacceptor site of intron 1 of the mouse Vκ3-7 gene. The reverse primerused for PCR of the human Vκ1-39 sequence included an extension encodinghuman Jκ5, whereas the reverse primer used for PCR of the human Vκ3-20sequence included an extension encoding human Jκ1. The human VpreBJλ5sequence was made by de novo DNA synthesis (Integrated DNATechnologies). A portion of the human Jκ-Cκ intron including the splicedonor site was PCR amplified from plasmid pBS-296-HA18-PIScel. Theforward PCR primer included an extension encoding part of either a humanJκ5, Jκ1, or Jκ5 sequence. The reverse primer included a PI-Scel site,which was previously engineered into the intron.

The mouse Vκ3-7 exon1/intron 1, human variable light chain exons, andhuman Jκ-Cκ intron fragments were sewn together by overlap extensionPCR, digested with Blpl and PI-Scel, and ligated into plasmidpBS-296-HA18-PIScel, which contained the promoter from the human Vκ3-15variable gene segment. A loxed hygromycin cassette within plasmidpBS-296-HA18-PIScel was replaced with a FRTed hygromycin cassetteflanked by NotI and AscI sites. The NotI/PI-Scel fragment of thisplasmid was ligated into modified mouse BAC 254m04, which contained partof the mouse Jκ-Cκ intron, the mouse CK exon, and about 75 kb of genomicsequence downstream of the mouse κ locus, which provided a 3′ homologyarm for homologous recombination in mouse ES cells. The Notl/Asclfragment of this BAC was then ligated into modified mouse BAC 302g12,which contained a FRTed neomycin cassette and about 23 kb of genomicsequence upstream of the endogenous κ locus for homologous recombinationin mouse ES cells.

Rearranged Human Germline Vκ1-39Jκ5 Targeting Vector (FIG. 1).Restriction enzyme sites were introduced at the 5′ and 3′ ends of anengineered light chain insert for cloning into a targeting vector: anAscl site at the 5′ end and a PI-Scel site at the 3′ end. Within the 5′AscI site and the 3′ PI-Scel site the targeting construct from 5′ to 3′included a 5′ homology arm containing sequence 5′ to the endogenousmouse κ light chain locus obtained from mouse BAC clone 302g12, a FRTedneomycin resistance gene, an genomic sequence including the human Vκ3-15promoter, a leader sequence of the mouse Vκ3-7 variable gene segment, aintron sequence of the mouse Vκ3-7 variable gene segment, an openreading frame of a rearranged human germline Vκ1-39Jκ5 region, a genomicsequence containing a portion of the human Jκ-Cκ intron, and a 3′homology arm containing sequence 3′ of the endogenous mouse Jκ5 genesegment obtained from mouse BAC clone 254m04 (FIG. 1, middle). Genesand/or sequences upstream of the endogenous mouse κ light chain locusand downstream of the most 3′ Jκ gene segment (e.g., the endogenous 3′enhancer) were unmodified by the targeting construct (see FIG. 1). Thesequence of the engineered human Vκ1-39Jκ5 locus is shown in SEQ IDNO:1.

Targeted insertion of the rearranged human germline Vκ1-39Jκ5 regioninto BAC DNA was confirmed by polymerase chain reaction (PCR) usingprimers located at sequences within the rearranged human germline lightchain region. Briefly, the intron sequence 3′ to the mouse Vκ3-7 leadersequence was confirmed with primers ULC-m1F (AGGTGAGGGT ACAGATAAGTGTTATGAG; SEQ ID NO:2) and ULC-m1R (TGACAAATGC CCTAATTATA GTGATCA; SEQID NO:3). The open reading frame of the rearranged human germlineVκ1-39Jκ5 region was confirmed with primers 1633-h2F (GGGCAAGTCAGAGCATTAGC A; SEQ ID NO:4) and 1633-h2R (TGCAAACTGG ATGCAGCATA G; SEQ IDNO:5). The neomycin cassette was confirmed with primers neoF (GGTGGAGAGGCTATTCGGC; SEQ ID NO:6) and neoR (GAACACGGCG GCATCAG; SEQ ID NO:7).Targeted BAC DNA was then used to electroporate mouse ES cells tocreated modified ES cells for generating chimeric mice that express arearranged human germline Vκ1-39Jκ5 region.

Positive ES cell clones were confirmed by TAQMAN™ screening andkaryotyping using probes specific for the engineered Vκ1-39Jκ5 lightchain region inserted into the endogenous locus. Briefly, probe neoP(TGGGCACAAC AGACAATCGG CTG; SEQ ID NO:8) which binds within the neomycinmarker gene, probe ULC-m1P (CCATTATGAT GCTCCATGCC TCTCTGTTC; SEQ IDNO:9) which binds within the intron sequence 3′ to the mouse Vκ3-7leader sequence, and probe 1633h2P (ATCAGCAGAA ACCAGGGAAA GCCCCT; SEQ IDNO:10) which binds within the rearranged human germline Vκ1-39Jκ5 openreading frame. Positive ES cell clones were then used to implant femalemice to give rise to a litter of pups expressing the germline Vκ1-39Jκ5light chain region.

Alternatively, ES cells bearing the rearranged human germline Vκ1-39Jκ5light chain region are transfected with a construct that expresses FLPin order to remove the FRTed neomycin cassette introduced by thetargeting construct. Optionally, the neomycin cassette is removed bybreeding to mice that express FLP recombinase (e.g., U.S. Pat. No.6,774,279). Optionally, the neomycin cassette is retained in the mice.

Rearranged Human Germline Vκ3-20Jκ1 Targeting Vector (FIG. 2). In asimilar fashion, an engineered light chain locus expressing a rearrangedhuman germline Vκ3-20Jκ1 region was made using a targeting constructincluding, from 5′ to 3′, a 5′ homology arm containing sequence 5′ tothe endogenous mouse κ light chain locus obtained from mouse BAC clone302g12, a FRTed neomycin resistance gene, a genomic sequence includingthe human Vκ3-15 promoter, a leader sequence of the mouse Vκ3-7 variablegene segment, an intron sequence of the mouse Vκ3-7 variable genesegment, an open reading frame of a rearranged human germline Vκ3-20Jκ1region, a genomic sequence containing a portion of the human Jκ-Cκintron, and a 3′ homology arm containing sequence 3′ of the endogenousmouse Jκ5 gene segment obtained from mouse BAC clone 254m04 (FIG. 2,middle). The sequence of the engineered human Vκ3-20Jκ1 locus is shownin SEQ ID NO:11.

Targeted insertion of the rearranged human germline Vκ3-20Jκ1 regioninto BAC DNA was confirmed by polymerase chain reaction (PCR) usingprimers located at sequences within the rearranged human germlineVκ3-20Jκ1 light chain region. Briefly, the intron sequence 3′ to themouse Vκ3-7 leader sequence was confirmed with primers ULC-m1F (SEQ IDNO:2) and ULC-m1R (SEQ ID NO:3). The open reading frame of therearranged human germline Vκ3-20Jκ1 region was confirmed with primers1635-h2F (TCCAGGCACC CTGTCTTTG; SEQ ID NO:12) and 1635-h2R (AAGTAGCTGCTGCTAACACT CTGACT; SEQ ID NO:13). The neomycin cassette was confirmedwith primers neoF (SEQ ID NO:6) and neoR (SEQ ID NO:7). Targeted BAC DNAwas then used to electroporate mouse ES cells to created modified EScells for generating chimeric mice that express the rearranged humangermline Vκ3-20Jκ1 light chain.

Positive ES cell clones were confirmed by TAQMAN™ screening andkaryotyping using probes specific for the engineered Vκ3-20Jκ1 lightchain region inserted into the endogenous κ light chain locus. Briefly,probe neoP (SEQ ID NO:8) which binds within the neomycin marker gene,probe ULC-m1P (SEQ ID NO:9) which binds within the mouse Vκ3-7 leadersequence, and probe 1635h2P (AAAGAGCCAC CCTCTCCTGC AGGG; SEQ ID NO:14)which binds within the human Vκ3-20Jκ1 open reading frame. Positive EScell clones were then used to implant female mice. A litter of pupsexpressing the human germline Vκ3-20Jκ1 light chain region.

Alternatively, ES cells bearing human germline Vκ3-20Jκ1 light chainregion can be transfected with a construct that expresses FLP in orderto remove the FRTed neomycin cassette introduced by the targetingconstruct. Optionally, the neomycin cassette may be removed by breedingto mice that express FLP recombinase (e.g., U.S. Pat. No. 6,774,279).Optionally, the neomycin cassette is retained in the mice.

Rearranged Human Germline VpreBJλ5 Targeting Vector (FIG. 3). In asimilar fashion, an engineered light chain locus expressing a rearrangedhuman germline VpreBJλ5 region was made using a targeting constructincluding, from 5′ to 3′, a 5′ homology arm containing sequence 5′ tothe endogenous mouse κ light chain locus obtained from mouse BAC clone302g12, a FRTed neomycin resistance gene, an genomic sequence includingthe human Vκ3-15 promoter, a leader sequence of the mouse Vκ3-7 variablegene segment, an intron sequence of the mouse Vκ3-7 variable genesegment, an open reading frame of a rearranged human germline VpreBJλ5region, a genomic sequence containing a portion of the human Jκ-Cκintron, and a 3′ homology arm containing sequence 3′ of the endogenousmouse Jκ5 gene segment obtained from mouse BAC clone 254m04 (FIG. 3,middle). The sequence of the engineered human VpreBJλ5 locus is shown inSEQ ID NO:15.

Targeted insertion of the rearranged human germline VpreBJλ5 region intoBAC DNA was confirmed by polymerase chain reaction (PCR) using primerslocated at sequences within the rearranged human germline VpreBJλ5region light chain region. Briefly, the intron sequence 3′ to the mouseVκ3-7 leader sequence was confirmed with primers ULC-m1F (SEQ ID NO:2)and ULC-m1R (SEQ ID NO:3). The open reading frame of the rearrangedhuman germline VpreBJλ5 region was confirmed with primers 1616-h1F(TGTCCTCGGC CCTTGGA; SEQ ID NO:16) and 1616-h1R (CCGATGTCAT GGTCGTTCCT;SEQ ID NO:17). The neomycin cassette was confirmed with primers neoF(SEQ ID NO:6) and neoR (SEQ ID NO:7). Targeted BAC DNA was then used toelectroporate mouse ES cells to created modified ES cells for generatingchimeric mice that express the rearranged human germline VpreBJλ5 lightchain.

Positive ES cell clones are confirmed by TAQMAN™ screening andkaryotyping using probes specific for the engineered VpreBJλ5 lightchain region inserted into the endogenous κ light chain locus. Briefly,probe neoP (SEQ ID NO:8) which binds within the neomycin marker gene,probe ULC-m1P (SEQ ID NO:9) which binds within the mouse IgVκ3-7 leadersequence, and probe 1616h1P (ACAATCCGCC TCACCTGCAC CCT; SEQ ID NO:18)which binds within the human VpreBJλ5 open reading frame. Positive EScell clones are then used to implant female mice to give rise to alitter of pups expressing a germline light chain region.

Alternatively, ES cells bearing the rearranged human germline VpreBJλ5light chain region are transfected with a construct that expresses FLPin order to remove the FRTed neomycin cassette introduced by thetargeting construct. Optionally, the neomycin cassette is removed bybreeding to mice that express FLP recombinase (e.g., U.S. Pat. No.6,774,279). Optionally, the neomycin cassette is retained in the mice.

Example 3 Generation of Mice Expressing a Single Rearranged Human LightChain

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0generation mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1):91-99. VELOCIMICE® independently bearing an engineeredhuman germline Vκ1-39Jκ5 light chain region, a Vκ3-20Jκ1 light chainregion or a VpreBJλ5 light chain region are identified by genotypingusing a modification of allele assay (Valenzuela et al., supra) thatdetects the presence of the unique rearranged human germline light chainregion.

Pups are genotyped and a pup heterozygous or homozygous for the uniquerearranged human germline light chain region are selected forcharacterizing expression of the rearranged human germline light chainregion.

Flow Cytometry. Expression of the rearranged human light chain region inthe normal antibody repertoire of common light chain mice was validatedby analysis of immunoglobulin κ and λ expression in splenocytes andperipheral blood of common light chain mice. Cell suspensions fromharvested spleens and peripheral blood of wild type (n=5), Vκ1-39Jκ5common light chain heterozygote (n=3), Vκ1-39Jκ5 common light chainhomozygote (n=3), Vκ3-20Jκ1 common light chain heterozygote (n=2), andVκ3-20Jκ1 common light chain homozygote (n=2) mice were made usingstandard methods and stained with CD19⁺, Igλ⁺ and Igκ⁺ usingfluorescently labeled antibodies (BD Pharmigen).

Briefly, 1×10⁶ cells were incubated with anti-mouse CD16/CD32 (clone2.4G2, BD Pharmigen) on ice for 10 minutes, followed by labeling withthe following antibody cocktail for 30 minutes on ice: APC conjugatedanti-mouse CD19 (clone 1D3, BD Pharmigen), PerCP-Cy5.5 conjugatedanti-mouse CD3 (clone 17A2, BioLegend), FITC conjugated anti-mouse Igκ(clone 187.1, BD Pharmigen), PE conjugated anti-mouse Igλ (clone RML-42,BioLegend). Following staining, cells were washed and fixed in 2%formaldehyde. Data acquisition was performed on an LSRII flow cytometerand analyzed with FlowJo. Gating: total B cells (CD19⁺CD3⁻), Igκ⁺ Bcells (Igκ⁺Igλ⁻CD19⁺CD3⁻), Igλ⁺ B cells (Igκ⁻Igλ⁺CD19⁺CD3⁻). Datagathered from blood and splenocyte samples demonstrated similar results.Table 3 sets forth the percent positive CD19⁺ B cells from peripheralblood of one representative mouse from each group that are Igλ⁺, Igκ⁺,or Igλ⁺Igκ⁺. Percent of CD19⁺ B cells in peripheral blood from wild type(WT) and mice homozygous for either the Vκ1-39Jκ5 or Vκ3-20Jκ1 commonlight chain are shown in FIG. 4.

TABLE 3 CD19⁺ B cells Mouse Igλ⁺ Igκ⁺ Igλ⁺Igκ⁺ wild type 4.8 93 0.53Vκ1-39Jκ5 1.4 93 2.6 Vκ3-20Jκ1 4.2 88 6

Common Light Chain Expression. Expression of each common light chain(Vκ1-39Jκ5 and Vκ3-20Jκ1) was analyzed in heterozygous and homozygousmice using a quantitative PCR assay (e.g. TAQMAN™).

Briefly, CD19⁺ B cells were purified from the spleens of wild type, micehomozygous for a replacement of the mouse heavy chain and κ light chainvariable region loci with corresponding human heavy chain and κ lightchain variable region loci (Hκ), as well as mice homozygous andheterozygous for each rearranged human light chain region (Vκ1-39Jκ5 orVκ3-20Jκ1) using mouse CD19 Microbeads (Miltenyi Biotec) according tomanufacturer's specifications. Total RNA was purified from CD19⁺ B cellsusing RNeasy Mini kit (Qiagen) according to manufacturer'sspecifications and genomic RNA was removed using an RNase-free DNaseon-column treatment (Qiagen). 200 ng mRNA was reverse-transcribed intocDNA using the First Stand cDNA Synthesis kit (Invitrogen) and theresulting cDNA was amplified with the Taqman Universal PCR Master Mix(Applied Biosystems). All reactions were performed using the ABI 7900Sequence Detection System (Applied Biosystems) using primers and TaqmanMGB probes spanning (1) the Vκ-Jκ junction for both common light chains,(2) the Vκ gene alone (i.e. Vκ1-39 and Vκ3-20), and (3) the mouse Cκregion. Table 4 sets forth the sequences of the primers and probesemployed for this assay. Relative expression was normalized toexpression of the mouse Cκ region. Results are shown in FIGS. 5A, 5B and5C.

TABLE 4 SEQ Primer/Probe Description  ID Region (5′-3′) NOs: Vκ1-39Jκ5(sense) 19 Junction AGCAGTCTGC AACCTGAAGA TTT (anti-sense) 20GTTTAATCTC CAGTCGTGTC CCTT (probe) 21 CCTCCGATCA CCTTC Vκ1-39 (sense) 22AAACCAGGGA AAGCCCCTAA (anti-sense) 23 ATGGGACCCC ACTTTGCA (probe) 24CTCCTGATCT ATGCTGCAT Vκ3-20Jκ1 (sense) 25 CAGCAGACTG GAGCCTGAAG AJunction (anti-sense) 26 TGATTTCCAC CTTGGTCCCT T (probe) 27TAGCTCACCT TGGACGTT Vκ3-20 (sense) 28 CTCCTCATCT ATGGTGCATC CA(anti-sense) 29 GACCCACTGC CACTGAACCT (probe) 30 CCACTGGCAT CCC Mouse Cκ(sense) 31 TGAGCAGCAC CCTCACGTT (anti-sense) 32GTGGCCTCAC AGGTATAGCT GTT (probe) 33 ACCAAGGACG AGTATGAA

Antigen Specific Common Light Chain Antibodies. Common light chain micebearing either a Vκ1-39Jκ5 or Vκ3-20Jκ1 common light chain at theendogenous mouse κlight chain locus were immunized with (3-galactosidaseand antibody titer was measured.

Briefly, β-galactosidase (Sigma) was emulsified in titermax adjuvant(Sigma), as per manufacturers directions. Wild type (n=7), Vκ1-39Jκ5common light chain homozygotes (n=2) and Vκ3-20Jκ1 common light chainhomozygotes (n=5) were immunized by subcutaneous injection with 100 μgβ-galactosidase/Titermax. Mice were boosted by subcutaneous injectiontwo times, 3 weeks apart, with 50 μg β-galactosidase/Titermax. After thesecond boost, blood was collected from anaesthetized mice using aretro-orbital bleed into serum separator tubes (BD Biosciences) as permanufacturer's directions. To measure anti-β-galactosidase IgM or IgGantibodies, ELISA plates (Nunc) were coated with 1 μg/mL β-galactosidaseovernight at 4° C. Excess antigen was washed off before blocking withPBS with 1% BSA for one hour at room temperature. Serial dilutions ofserum were added to the plates and incubated for one hour at roomtemperature before washing. Plates were then incubated with HRPconjugated anti-IgM (Southern Biotech) or anti-IgG (Southern Biotech)for one hour at room temperature. Following another wash, plates weredeveloped with TMB substrate (BD Biosciences). Reactions were stoppedwith 1N sulfuric acid and OD₄₅₀ was read using a Victor X5 Plate Reader(Perkin Elmer). Data was analyzed with GraphPad Prism and signal wascalculated as the dilution of serum that is two times above background.Results are shown in FIGS. 6A and 6B.

As shown in this Example, the ratio of κ/λ B cells in both the splenicand peripheral compartments of Vκ1-39Jκ5 and Vκ3-20Jκ1 common lightchain mice demonstrated a near wild type pattern (Table 3 and FIG. 4).VpreBJλ5 common light chain mice, however, demonstrated fewer peripheralB cells, of which about 1-2% express the engineered human light chainregion (data not shown). The expression levels of the Vκ1-39Jκ5 andVκ3-20Jκ1 rearranged human light chain regions from the endogenous κlight chain locus were elevated in comparison to an endogenous κ lightchain locus containing a complete replacement of mouse Vκ and Jκ genesegments with human Vκ and Jκ gene segments (FIGS. 5A, 5B and 5C). Theexpression levels of the VpreBJλ5 rearranged human light chain regiondemonstrated similar high expression from the endogenous κ light chainlocus in both heterozygous and homozygous mice (data not shown). Thisdemonstrates that in direct competition with the mouse λ, κ, or bothendogenous light chain loci, a single rearranged human V_(L)/J_(L)sequence can yield better than wild type level expression from theendogenous κ light chain locus and give rise to normal splenic and bloodB cell frequency. Further, the presence of an engineered κ light chainlocus having either a human Vκ1-39Jκ5 or human Vκ3-20Jκ1 sequence waswell tolerated by the mice and appear to function in wild type fashionby representing a substantial portion of the light chain repertoire inthe humoral component of the immune response (FIGS. 6A and 6B).

Example 4 Breeding of Mice Expressing a Single Rearranged Human GermlineLight Chain

This Example describes several other genetically modified mouse strainsthat can be bred to any one of the common light chain mice describedherein to create multiple genetically modified mouse strains harboringmultiple genetically modified immunoglobulin loci.

Endogenous Igλ Knockout (KO). To optimize the usage of the engineeredlight chain locus, mice bearing one of the rearranged human germlinelight chain regions are bred to another mouse containing a deletion inthe endogenous λ light chain locus. In this manner, the progeny obtainedwill express, as their only light chain, the rearranged human germlinelight chain region as described in Example 2. Breeding is performed bystandard techniques recognized in the art and, alternatively, by acommercial breeder (e.g., The Jackson Laboratory). Mouse strains bearingan engineered light chain locus and a deletion of the endogenous λ lightchain locus are screened for presence of the unique light chain regionand absence of endogenous mouse λ light chains.

Humanized Endogenous Heavy Chain Locus. Mice bearing an engineered humangermline light chain locus are bred with mice that contain a replacementof the endogenous mouse heavy chain variable gene locus with the humanheavy chain variable gene locus (see U.S. Pat. No. 6,596,541; theVELOCIMMUNE® mouse, Regeneron Pharmaceuticals, Inc.). The VELOCIMMUNE®mouse comprises a genome comprising human heavy chain variable regionsoperably linked to endogenous mouse constant region loci such that themouse produces antibodies comprising a human heavy chain variable regionand a mouse heavy chain constant region in response to antigenicstimulation. The DNA encoding the variable regions of the heavy chainsof the antibodies is isolated and operably linked to DNA encoding thehuman heavy chain constant regions. The DNA is then expressed in a cellcapable of expressing the fully human heavy chain of the antibody.

Mice bearing a replacement of the endogenous mouse V_(H) locus with thehuman VH locus and a single rearranged human germline V_(L) region atthe endogenous κ light chain locus are obtained. Reverse chimericantibodies containing somatically mutated heavy chains (human V_(H) andmouse C_(H)) with a single human light chain (human V_(L) and mouseC_(L)) are obtained upon immunization with an antigen of interest. V_(H)and V_(L) nucleotide sequences of B cells expressing the antibodies areidentified and fully human antibodies are made by fusion the V_(H) andV_(L) nucleotide sequences to human C_(H) and C_(L) nucleotide sequencesin a suitable expression system.

Example 5 Generation of Antibodies from Mice Expressing Human HeavyChains and a Rearranged Human Germline Light Chain Region

After breeding mice that contain the engineered human light chain regionto various desired strains containing modifications and deletions ofother endogenous Ig loci (as described in Example 4), selected mice canbe immunized with an antigen of interest.

Generally, a VELOCIMMUNE® mouse containing one of the single rearrangedhuman germline light chain regions is challenged with an antigen, andlymphatic cells (such as B-cells) are recovered from serum of theanimals. The lymphatic cells are fused with a myeloma cell line toprepare immortal hybridoma cell lines, and such hybridoma cell lines arescreened and selected to identify hybridoma cell lines that produceantibodies containing human heavy chain variables and a rearranged humangermline light chains which are specific to the antigen used forimmunization. DNA encoding the variable regions of the heavy chains andthe light chain are isolated and linked to desirable isotypic constantregions of the heavy chain and light chain. Due to the presence of theendogenous mouse sequences and any additional cis-acting elementspresent in the endogenous locus, the single light chain of each antibodymay be somatically mutated. This adds additional diversity to theantigen-specific repertoire comprising a single light chain and diverseheavy chain sequences. The resulting cloned antibody sequences aresubsequently expressed in a cell, such as a CHO cell. Alternatively, DNAencoding the antigen-specific chimeric antibodies or the variabledomains of the light and heavy chains are identified directly fromantigen-specific lymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. As described above, theantibodies are characterized and selected for desirable characteristics,including affinity, selectivity, epitope, etc. The mouse constantregions are replaced with a desired human constant region to generatethe fully human antibody containing a somatically mutated human heavychain and a single light chain derived from a rearranged human germlinelight chain region of the invention. Suitable human constant regionsinclude, for example wild type or modified IgG1 or IgG4.

Separate cohorts of VELOCIMMUNE® mice containing a replacement of theendogenous mouse heavy chain locus with human V_(H), D_(H), and J_(H)gene segments and a replacement of the endogenous mouse κ light chainlocus with either the engineered germline Vκ1-39Jκ5 human light chainregion or the engineered germline Vκ3-20Jκ1 human light chain region(described above) were immunized with a human cell-surface receptorprotein (Antigen E). Antigen E is administered directly onto the hindfootpad of mice with six consecutive injections every 3-4 days. Two tothree micrograms of Antigen E are mixed with 10 μg of CpGoligonucleotide (Cat # tlrl-modn—ODN1826 oligonucleotide; InVivogen, SanDiego, Calif.) and 25 μg of Adju-Phos (Aluminum phosphate gel adjuvant,Cat# H-71639-250; Brenntag Biosector, Frederikssund, Denmark) prior toinjection. A total of six injections are given prior to the finalantigen recall, which is given 3-5 days prior to sacrifice. Bleeds afterthe 4th and 6th injection are collected and the antibody immune responseis monitored by a standard antigen-specific immunoassay.

When a desired immune response is achieved splenocytes are harvested andfused with mouse myeloma cells to preserve their viability and formhybridoma cell lines. The hybridoma cell lines are screened and selectedto identify cell lines that produce Antigen E-specific common lightchain antibodies. Using this technique several anti-Antigen E-specificcommon light chain antibodies (i.e., antibodies possessing human heavychain variable domains, the same human light chain variable domain, andmouse constant domains) are obtained.

Alternatively, anti-Antigen E common light chain antibodies are isolateddirectly from antigen-positive B cells without fusion to myeloma cells,as described in U.S. 2007/0280945A1, herein specifically incorporated byreference in its entirety. Using this method, several fully humananti-Antigen E common light chain antibodies (i.e., antibodiespossessing human heavy chain variable domains, either an engineeredhuman Vκ1-39Jκ5 light chain or an engineered human Vκ3-20Jκ1 light chainregion, and human constant domains) were obtained.

The biological properties of the exemplary anti-Antigen E common lightchain antibodies generated in accordance with the methods of thisExample are described in detail in the sections set forth below.

Example 6 Heavy Chain Gene Segment Usage in Antigen-Specific CommonLight Chain Antibodies

To analyze the structure of the human anti-Antigen E common light chainantibodies produced, nucleic acids encoding heavy chain antibodyvariable regions were cloned and sequenced. From the nucleic acidsequences and predicted amino acid sequences of the antibodies, geneusage was identified for the heavy chain variable region (HCVR) ofselected common light chain antibodies obtained from immunizedVELOCIMMUNE® mice containing either the engineered human Vκ1-39Jκ5 lightchain or engineered human Vκ3-20Jκ1 light chain region. Results areshown in Tables 5 and 6, which demonstrate that mice according to theinvention generate antigen-specific common light chain antibodies from avariety of human heavy chain gene segments, due to a variety ofrearrangements, when employing either a mouse that expresses a lightchain from only a human Vκ1-39- or a human Vκ3-20-derived light chain.Human V_(H) gene segments of the 2, 3, 4, and 5 families rearranged witha variety of human D_(H) segments and human J_(H) segments to yieldantigen-specific antibodies.

TABLE 5 Vκ1-39Jκ5 Common Light Chain Antibodies HCVR Antibody V_(H)D_(H) J_(H) 2952 2-5  6-6  1 5978 2-5  6-6  1 5981 2-5  3-22 1 6027 3-136-6  5 3022 3-23 3-10 4 3028 3-23 3-3  4 5999 3-23 6-6  4 6009 3-23 2-8 4 6011 3-23 7-27 4 5980 3-30 1-1  4 3014 3-30 1-7  4 3015 3-30 1-7  43023 3-30 1-7  4 3024 3-30 1-7  4 3032 3-30 1-7  4 6024 3-30 1-7  4 60253-30 1-7  4 6031 3-30 1-7  4 6007 3-30 3-3  4 2982 3-30 3-22 5 6001 3-303-22 5 6005 3-30 3-22 5 6035 3-30 5-5  2 3013 3-30 5-12 4 3042 3-30 5-124 2955 3-30 6-6  1 3043 3-30 6-6  3 3018 3-30 6-6  4 2949 3-30 6-6  52950 3-30 6-6  5 2954 3-30 6-6  5 2978 3-30 6-6  5 3016 3-30 6-6  5 30173-30 6-6  5 3033 3-30 6-6  5 3041 3-30 6-6  5 5979 3-30 6-6  5 5998 3-306-6  5 6004 3-30 6-6  5 6010 3-30 6-6  5 6019 3-30 6-6  5 6021 3-30 6-6 5 6022 3-30 6-6  5 6023 3-30 6-6  5 6030 3-30 6-6  5 6032 3-30 6-6  52985 3-30 6-13 4 2997 3-30 6-13 4 3011 3-30 6-13 4 3047 3-30 6-13 4 59823-30 6-13 4 6002 3-30 6-13 4 6003 3-30 6-13 4 6012 3-30 6-13 4 6013 3-306-13 4 6014 3-30 6-13 4 6015 3-30 6-13 4 6016 3-30 6-13 4 6017 3-30 6-134 6020 3-30 6-13 4 6034 3-30 6-13 4 2948 3-30 7-27 4 2987 3-30 7-27 42996 3-30 7-27 4 3005 3-30 7-27 4 3012 3-30 7-27 4 3020 3-30 7-27 4 30213-30 7-27 4 3025 3-30 7-27 4 3030 3-30 7-27 4 3036 3-30 7-27 4 5997 3-307-27 4 6033 3-30 7-27 4 3004 3-30 7-27 5 6028 3-30 7-27 6 3010 4-59 3-163 3019 4-59 3-16 3 6018 4-59 3-16 3 6026 4-59 3-16 3 6029 4-59 3-16 36036 4-59 3-16 3 6037 4-59 3-16 3 2964 4-59 3-22 3 3027 4-59 3-16 4 30465-51 5-5  3 6000 1-69 6-13 4 6006 1-69 6-6  5 6008 1-69 6-13 4

TABLE 6 Vκ3-20Jκ1 Common Light Chain Antibodies HCVR Antibody V_(H)D_(H) J_(H) 5989 3-30 3-3  3 5994 3-33 1-7  4 5985 3-33 2-15 4 5987 3-332-15 4 5995 3-33 2-15 4 2968 4-39 1-26 3 5988 4-39 1-26 3 5990 4-39 1-263 5992 4-39 1-26 3 2975 5-51 6-13 5 2972 5-51 3-16 6 5986 5-51 3-16 65993 5-51 3-16 6 5996 5-51 3-16 6 5984 3-53 1-1  4

Example 7 Determination of Blocking Ability of Antigen-Specific CommonLight Chain Antibodies by LUMINEX™ Assay

Ninety-eight human common light chain antibodies raised against AntigenE were tested for their ability to block binding of Antigen E's naturalligand (Ligand Y) to Antigen E in a bead-based assay.

The extracellular domain (ECD) of Antigen E was conjugated to two mycepitope tags and a 6× histidine tag (Antigen E-mmH) and amine-coupled tocarboxylated microspheres at a concentration of 20 μg/mL in MES buffer.The mixture was incubated for two hours at room temperature followed bybead deactivation with 1M Tris pH 8.0 followed by washing in PBS with0.05% (v/v) Tween-20. The beads were then blocked with PBS (IrvineScientific, Santa Ana, Calif.) containing 2% (w/v) BSA (Sigma-AldrichCorp., St. Louis, Mo.). In a 96-well filter plate, supernatantscontaining Antigen E-specific common light chain antibodies, werediluted 1:15 in buffer. A negative control containing a mock supernatantwith the same media components as for the antibody supernatant wasprepared. Antigen E-labeled beads were added to the supernatants andincubated overnight at 4° C. Biotinylated-Ligand Y protein was added toa final concentration of 0.06 nM and incubated for two hours at roomtemperature. Detection of biotinylated-Ligand Y bound to AntigenE-myc-myc-6His labeled beads was determined with R-Phycoerythrinconjugated to Streptavidin (Moss Inc, Pasadena, Md.) followed bymeasurement in a LUMINEX™ flow cytometry-based analyzer. Background MeanFluorescence Intensity (MFI) of a sample without Ligand Y was subtractedfrom all samples. Percent blocking was calculated by division of thebackground-subtracted MFI of each sample by the adjusted negativecontrol value, multiplying by 100 and subtracting the resulting valuefrom 100.

In a similar experiment, the same 98 human common light chain antibodiesraised against Antigen E were tested for their ability to block bindingof Antigen E to Ligand Y-labeled beads.

Briefly, Ligand Y was amine-coupled to carboxylated microspheres at aconcentration of 20 μg/mL diluted in MES buffer. The mixture andincubated two hours at room temperature followed by deactivation ofbeads with 1M Tris pH 8 then washing in PBS with 0.05% (v/v) Tween-20.The beads were then blocked with PBS (Irvine Scientific, Santa Ana,Calif.) containing 2% (w/v) BSA (Sigma-Aldrich Corp., St. Louis, Mo.).In a 96-well filter plate, supernatants containing Antigen E-specificcommon light chain antibodies were diluted 1:15 in buffer. A negativecontrol containing a mock supernatant with the same media components asfor the antibody supernatant was prepared. A biotinylated-Antigen E-mmHwas added to a final concentration of 0.42 nM and incubated overnight at4° C. Ligand Y-labeled beads were then added to the antibody/Antigen Emixture and incubated for two hours at room temperature. Detection ofbiotinylated-Antigen E-mmH bound to Ligand Y-beads was determined withR-Phycoerythrin conjugated to Streptavidin (Moss Inc, Pasadena, Md.)followed by measurement in a LUMINEX™ flow cytometry-based analyzer.Background Mean Fluorescence Intensity (MFI) of a sample without AntigenE was subtracted from all samples. Percent blocking was calculated bydivision of the background-subtracted MFI of each sample by the adjustednegative control value, multiplying by 100 and subtracting the resultingvalue from 100.

Tables 7 and 8 show the percent blocking for all 98 anti-Antigen Ecommon light chain antibodies tested in both LUMINEX™ assays. ND: notdetermined under current experimental conditions.

TABLE 7 Vκ1-39Jκ5 Common Light Chain Antibodies % Blocking of % Blockingof Antibody Antigen E-Labeled Beads Antigen E In Solution 2948 81.1 47.82948G 38.6 ND 2949 97.6 78.8 2949G 97.1 73.7 2950 96.2 81.9 2950G 89.831.4 2952 96.1 74.3 2952G 93.5 39.9 2954 93.7 70.1 2954G 91.7 30.1 295575.8 30.0 2955G 71.8 ND 2964 92.1 31.4 2964G 94.6 43.0 2978 98.0 95.12978G 13.9 94.1 2982 92.8 78.5 2982G 41.9 52.4 2985 39.5 31.2 2985G 2.05.0 2987 81.7 67.8 2987G 26.6 29.3 2996 87.3 55.3 2996G 95.9 38.4 299793.4 70.6 2997G 9.7 7.5 3004 79.0 48.4 3004G 60.3 40.7 3005 97.4 93.53005G 77.5 75.6 3010 98.0 82.6 3010G 97.9 81.0 3011 87.4 42.8 3011G 83.541.7 3012 91.0 60.8 3012G 52.4 16.8 3013 80.3 65.8 3013G 17.5 15.4 301463.4 20.7 3014G 74.4 28.5 3015 89.1 55.7 3015G 58.8 17.3 3016 97.1 81.63016G 93.1 66.4 3017 94.8 70.2 3017G 87.9 40.8 3018 85.4 54.0 3018G 26.112.7 3019 99.3 92.4 3019G 99.3 88.1 3020 96.7 90.3 3020G 85.2 41.5 302174.5 26.1 3021G 81.1 27.4 3022 65.2 17.6 3022G 67.2 9.1 3023 71.4 28.53023G 73.8 29.7 3024 73.9 32.6 3024G 89.0 10.0 3025 70.7 15.6 3025G 76.724.3 3027 96.2 61.6 3027G 98.6 75.3 3028 92.4 29.0 3028G 87.3 28.8 30306.0 10.6 3030G 41.3 14.2 3032 76.5 31.4 3032G 17.7 11.0 3033 98.2 86.13033G 93.6 64.0 3036 74.7 32.7 3036G 90.1 51.2 3041 95.3 75.9 3041G 92.451.6 3042 88.1 73.3 3042G 60.9 25.2 3043 90.8 65.8 3043G 92.8 60.3

TABLE 8 Vκ3-20Jκ1 Common Light Chain Antibodies % Blocking of % Blockingof Antibody Antigen E-Labeled Beads Antigen E In Solution 2968 97.1 73.32968G 67.1 14.6 2969 51.7 20.3 2969G 37.2 16.5 2970 92.2 34.2 2970G 92.727.2 2971 23.4 11.6 2971G 18.8 18.9 2972 67.1 38.8 2972G 64.5 39.2 297377.7 27.0 2973G 51.1 20.7 2974 57.8 12.4 2974G 69.9 17.6 2975 49.4 18.22975G 32.0 19.5 2976 1.0 1.0 2976G 50.4 20.4

In the first LUMINEX™ experiment described above, 80 common light chainantibodies containing the Vκ1-39Jκ5 engineered light chain were testedfor their ability to block Ligand Y binding to Antigen E-labeled beads.Of these 80 common light chain antibodies, 68 demonstrated>50% blocking,while 12 demonstrated<50% blocking (6 at 25-50% blocking and 6 at <25%blocking). For the 18 common light chain antibodies containing theVκ3-20Jκ1 engineered light chain, 12 demonstrated>50% blocking, while 6demonstrated<50% blocking (3 at 25-50% blocking and 3 at <25% blocking)of Ligand Y binding to Antigen E-labeled beads.

In the second LUMINEX™ experiment described above, the same 80 commonlight chain antibodies containing the Vκ1-39Jκ5 engineered light chainwere tested for their ability to block binding of Antigen E to LigandY-labeled beads. Of these 80 common light chain antibodies, 36demonstrated>50% blocking, while 44 demonstrated<50% blocking (27 at25-50% blocking and 17 at <25% blocking). For the 18 common light chainantibodies containing the Vκ3-20Jκ1 engineered light chain, 1demonstrated>50% blocking, while 17 demonstrated<50% blocking (5 at25-50% blocking and 12 at <25% blocking) of Antigen E binding to LigandY-labeled beads.

The data of Tables 7 and 8 establish that the rearrangements describedin Tables 5 and 6 generated anti-Antigen E-specific common light chainantibodies that blocked binding of Ligand Y to its cognate receptorAntigen E with varying degrees of efficacy, which is consistent with theanti-Antigen E common light chain antibodies of Tables 5 and 6comprising antibodies with overlapping and non-overlapping epitopespecificity with respect to Antigen E.

Example 8 Determination of Blocking Ability of Antigen-Specific CommonLight Chain Antibodies by ELISA

Human common light chain antibodies raised against Antigen E were testedfor their ability to block Antigen E binding to a Ligand Y-coatedsurface in an ELISA assay.

Ligand Y was coated onto 96-well plates at a concentration of 2 pg/mLdiluted in PBS and incubated overnight followed by washing four times inPBS with 0.05% Tween-20. The plate was then blocked with PBS (IrvineScientific, Santa Ana, Calif.) containing 0.5% (w/v) BSA (Sigma-AldrichCorp., St. Louis, Mo.) for one hour at room temperature. In a separateplate, supernatants containing anti-Antigen E common light chainantibodies were diluted 1:10 in buffer. A mock supernatant with the samecomponents of the antibodies was used as a negative control. AntigenE-mmH (described above) was added to a final concentration of 0.150 nMand incubated for one hour at room temperature. The antibody/AntigenE-mmH mixture was then added to the plate containing Ligand Y andincubated for one hour at room temperature. Detection of Antigen E-mmHbound to Ligand Y was determined with Horse-Radish Peroxidase (HRP)conjugated to anti-Penta-His antibody (Qiagen, Valencia, Calif.) anddeveloped by standard colorimetric response using tetramethylbenzidine(TMB) substrate (BD Biosciences, San Jose, Calif.) neutralized bysulfuric acid. Absorbance was read at OD450 for 0.1 sec. Backgroundabsorbance of a sample without Antigen E was subtracted from allsamples. Percent blocking was calculated by division of thebackground-subtracted MFI of each sample by the adjusted negativecontrol value, multiplying by 100 and subtracting the resulting valuefrom 100.

Tables 9 and 10 show the percent blocking for all 98 anti-Antigen Ecommon light chain antibodies tested in the ELISA assay. ND: notdetermined under current experimental conditions.

TABLE 9 Vκ1-39Jκ5 Common Light Chain Antibodies % Blocking of AntibodyAntigen E In Solution 2948 21.8 2948G 22.9 2949 79.5 2949G 71.5 295080.4 2950G 30.9 2952 66.9 2952G 47.3 2954 55.9 2954G 44.7 2955 12.12955G 25.6 2964 34.8 2964G 47.7 2978 90.0 2978G 90.2 2982 59.0 2982G20.4 2985 10.5 2985G ND 2987 31.4 2987G ND 2996 29.3 2996G ND 2997 48.72997G ND 3004 16.7 3004G 3.5 3005 87.2 3005G 54.3 3010 74.5 3010G 84.63011 19.4 3011G ND 3012 45.0 3012G 12.6 3013 39.0 3013G 9.6 3014 5.23014G 17.1 3015 23.7 3015G 10.2 3016 78.1 3016G 37.4 3017 61.6 3017G25.2 3018 40.6 3018G 14.5 3019 94.6 3019G 92.3 3020 80.8 3020G ND 30217.6 3021G 20.7 3022 2.4 3022G 15.0 3023 9.1 3023G 19.2 3024 7.5 3024G15.2 3025 ND 3025G 13.9 3027 61.4 3027G 82.7 3028 40.3 3028G 12.3 3030ND 3030G 9.5 3032 ND 3032G 13.1 3033 77.1 3033G 32.9 3036 17.6 3036G24.6 3041 59.3 3041G 30.7 3042 39.9 3042G 16.1 3043 57.4 3043G 46.1

TABLE 10 Vκ3-20Jκ1 Common Light Chain Antibodies % Blocking of AntibodyAntigen E In Solution 2968 68.9 2968G 15.2 2969 10.1 2969G 23.6 297034.3 2970G 41.3 2971 6.3 2971G 27.1 2972 9.6 2972G 35.7 2973 20.7 2973G23.1 2974 ND 2974G 22.0 2975 8.7 2975G 19.2 2976 4.6 2976G 26.7

As described in this Example, of the 80 common light chain antibodiescontaining the Vκ1-39Jκ5 engineered light chain tested for their abilityto block Antigen E binding to a Ligand Y-coated surface, 22demonstrated>50% blocking, while 58 demonstrated<50% blocking (20 at25-50% blocking and 38 at <25% blocking). For the 18 common light chainantibodies containing the Vκ3-20Jκ1 engineered light chain, onedemonstrated>50% blocking, while 17 demonstrated<50% blocking (5 at25-50% blocking and 12 at <25% blocking) of Antigen E binding to aLigand Y-coated surface.

These results are also consistent with the Antigen E-specific commonlight chain antibody pool comprising antibodies with overlapping andnon-overlapping epitope specificity with respect to Antigen E.

Example 9 BIACORE™ Affinity Determination for Antigen-Specific CommonLight Chain Antibodies

Equilibrium dissociation constants (K_(D)) for selected antibodysupernatants were determined by SPR (Surface Plasmon Resonance) using aBIACORE™ T100 instrument (GE Healthcare). All data was obtained usingHBS-EP (10mM Hepes, 150 mM NaCl, 0.3 mM EDTA, 0.05% Surfactant P20, pH7.4) as both the running and sample buffers, at 25° C. Antibodies werecaptured from crude supernatant samples on a CM5 sensor chip surfacepreviously derivatized with a high density of anti-human Fc antibodiesusing standard amine coupling chemistry. During the capture step,supernatants were injected across the anti-human Fc surface at a flowrate of 3 μL/min, for a total of 3 minutes. The capture step wasfollowed by an injection of either running buffer or analyte at aconcentration of 100 nM for 2 minutes at a flow rate of 35 μL/min.Dissociation of antigen from the captured antibody was monitored for 6minutes. The captured antibody was removed by a brief injection of 10 mMglycine, pH 1.5. All sensorgrams were double referenced by subtractingsensorgrams from buffer injections from the analyte sensorgrams, therebyremoving artifacts caused by dissociation of the antibody from thecapture surface. Binding data for each antibody was fit to a 1:1 bindingmodel with mass transport using BIAcore T100 Evaluation software v2.1.Results are shown in Tables 11 and 12.

TABLE 11 Vκ1-39Jκ5 Common Light Chain Antibodies 100 nM Antigen EAntibody K_(D) (nM) T_(1/2) (min) 2948 8.83 28 2948G 95.0 1 2949 3.57 182949G 6.37 9 2950 4.91 17 2950G 13.6 5 2952 6.25 7 2952G 7.16 4 29542.37 24 2954G 5.30 9 2955 14.4 6 2955G 12.0 4 2964 14.8 6 2964G 13.0 92978 1.91 49 2978G 1.80 58 2982 6.41 19 2982G 16.3 9 2985 64.4 9 2985G2.44 8 2987 21.0 11 2987G 37.6 4 2996 10.8 9 2996G 24.0 2 2997 7.75 192997G 151 1 3004 46.5 14 3004G 1.93 91 3005 2.35 108 3005G 6.96 27 30104.13 26 3010G 2.10 49 3011 59.1 5 3011G 41.7 5 3012 9.71 20 3012G 89.9 23013 20.2 20 3013G 13.2 4 3014 213 4 3014G 36.8 3 3015 29.1 11 3015G65.9 0 3016 4.99 17 3016G 18.9 4 3017 9.83 8 3017G 55.4 2 3018 11.3 363018G 32.5 3 3019 1.54 59 3019G 2.29 42 3020 5.41 39 3020G 41.9 6 302150.1 6 3021G 26.8 4 3022 25.7 17 3022G 20.8 12 3023 263 9 3023G 103 53024 58.8 7 3024G 7.09 10 3025 35.2 6 3025G 42.5 8 3027 7.15 6 3027G4.24 18 3028 6.89 37 3028G 7.23 22 3030 46.2 7 3030G 128 3 3032 53.2 93032G 13.0 1 3033 4.61 17 3033G 12.0 5 3036 284 12 3036G 18.2 10 30416.90 12 3041G 22.9 2 3042 9.46 34 3042G 85.5 3 3043 9.26 29 3043G 13.122

TABLE 12 Vκ3-20Jκ1 Common Light Chain Antibodies 100 nM Antigen EAntibody K_(D) (nM) T_(1/2) (min) 2968 5.50 8 2968G 305 0 2969 34.9 22969G 181 1 2970G 12.3 3 2971G 32.8 22 2972 6.02 13 2972G 74.6 26 29735.35 39 2973G 11.0 44 2974 256 0 2974G 138 0 2975 38.0 2 2975G 134 12976 6.73 10 2976G 656 8

The binding affinities of common light chain antibodies comprising therearrangements shown in Tables 5 and 6 vary, with nearly all exhibitinga K_(D) in the nanomolar range. The affinity data is consistent with thecommon light chain antibodies resulting from the combinatorialassociation of rearranged variable domains described in Tables 5 and 6being high-affinity, clonally selected, and somatically mutated. Coupledwith data previously shown, the common light chain antibodies describedin Tables 5 and 6 comprise a collection of diverse, high-affinityantibodies that exhibit specificity for one or more epitopes on AntigenE.

Example 10 Determination of Binding Specificities of Antigen-SpecificCommon Light Chain Antibodies by LUMINEX™ Assay

Selected anti-Antigen E common light chain antibodies were tested fortheir ability to bind to the ECD of Antigen E and Antigen E ECDvariants, including the cynomolgous monkey ortholog (Mf Antigen E),which differs from the human protein in approximately 10% of its aminoacid residues; a deletion mutant of Antigen E lacking the last 10 aminoacids from the C-terminal end of the ECD (Antigen E-ACT); and twomutants containing an alanine substitution at suspected locations ofinteraction with Ligand Y (Antigen E-Ala1 and AntigenE-Ala2). TheAntigen E proteins were produced in CHO cells and each contained amyc-myc-His C-terminal tag.

For the binding studies, Antigen E ECD protein or variant protein(described above) from 1 mL of culture medium was captured by incubationfor 2 hr at room temperature with 1×10⁶ microsphere (LUMINEX™) beadscovalently coated with an anti-myc monoclonal antibody (MAb 9E10,hybridoma cell line CRL-1729™; ATCC, Manassas, Va.). The beads were thenwashed with PBS before use. Supernatants containing anti-Antigen Ecommon light chain antibodies were diluted 1:4 in buffer and added to96-well filter plates. A mock supernatant with no antibody was used asnegative control. The beads containing the captured Antigen E proteinswere then added to the antibody samples (3000 beads per well) andincubated overnight at 4° C. The following day, the sample beads werewashed and the bound common light chain antibody was detected with aR-phycoerythrin-conjugated anti-human IgG antibody. The fluorescenceintensity of the beads (approximately 100 beads counted for eachantibody sample binding to each Antigen E protein) was measured with aLUMINEX™ flow cytometry-based analyzer, and the median fluorescenceintensity (MFI) for at least 100 counted beads per bead/antibodyinteraction was recorded. Results are shown in Tables 13 and 14.

TABLE 13 Vκ1-39Jκ5 Common Light Chain Antibodies Mean FluorescenceIntensity (MFI) Antigen Antigen Antigen E- Antigen E- Mf Antibody E-ECDE-ΔCT Ala1 Ala2 Antigen E 2948 1503 2746 4953 3579 1648 2948G 537 6622581 2150 863 2949 3706 4345 8169 5678 5142 2949G 3403 3318 7918 58265514 2950 3296 4292 7756 5171 4749 2950G 2521 2408 7532 5079 3455 29523384 1619 1269 168 911 2952G 3358 1001 108 55 244 2954 2808 3815 71145039 3396 2954G 2643 2711 7620 5406 3499 2955 1310 2472 4738 3765 16372955G 1324 1802 4910 3755 1623 2964 5108 1125 4185 346 44 2964G 4999 7294646 534 91 2978 6986 2800 14542 10674 8049 2978G 5464 3295 11652 80266452 2982 4955 2388 13200 9490 6772 2982G 3222 2013 8672 6509 4949 29851358 832 4986 3892 1669 2985G 43 43 128 244 116 2987 3117 1674 7646 59442546 2987G 3068 1537 9202 6004 4744 2996 4666 1917 12875 9046 6459 2996G2752 1736 8742 6150 4873 2997 5164 2159 12167 8361 5922 2997G 658 3563392 2325 1020 3004 2794 1397 8542 6268 3083 3004G 2753 1508 8267 58084345 3005 5683 2221 12900 9864 5868 3005G 4344 2732 10669 7125 5880 30104829 1617 2642 3887 44 3010G 3685 1097 2540 3022 51 3011 2859 2015 78555513 3863 3011G 2005 1072 6194 4041 3181 3012 3233 2221 8543 5637 33073012G 968 378 3115 2261 1198 3013 2343 1791 6715 4810 2528 3013G 327 1441333 1225 370 3014 1225 1089 5436 3621 1718 3014G 1585 851 5178 37052411 3015 3202 2068 8262 5554 3796 3015G 1243 531 4246 2643 1611 30164220 2543 8920 5999 5666 3016G 2519 1277 6344 4288 4091 3017 3545 25538700 5547 5098 3017G 1972 1081 5763 3825 3038 3018 2339 1971 6140 45152293 3018G 254 118 978 1020 345 3019 5235 1882 7108 4249 54 3019G 40901270 4769 3474 214 3020 3883 3107 8591 6602 4420 3020G 2165 1209 64894295 2912 3021 1961 1472 6872 4641 2742 3021G 2091 1005 6430 3988 29353022 2418 793 7523 2679 36 3022G 2189 831 6182 3051 132 3023 1692 14115788 3898 2054 3023G 1770 825 5702 3677 2648 3024 1819 1467 6179 45572450 3024G 100 87 268 433 131 3025 1853 1233 6413 4337 2581 3025G 1782791 5773 3871 2717 3027 4131 1018 582 2510 22 3027G 3492 814 1933 259642 3028 4361 2545 9884 5639 975 3028G 2835 1398 7124 3885 597 3030 463277 1266 1130 391 3030G 943 302 3420 2570 1186 3032 2083 1496 6594 44022405 3032G 295 106 814 902 292 3033 4409 2774 8971 6331 5825 3033G 24991234 6745 4174 4210 3036 1755 1362 6137 4041 1987 3036G 2313 1073 63874243 3173 3041 3674 2655 8629 5837 4082 3041G 2519 1265 6468 4274 33203042 2653 2137 7277 5124 3325 3042G 1117 463 4205 2762 1519 3043 30362128 7607 5532 3366 3043G 2293 1319 6573 4403 3228

TABLE 14 Vκ3-20Jκ1 Common Light Chain Antibodies Mean FluorescenceIntensity (MFI) Antigen Antigen Antigen E- Antigen E- Mf AntigenAntibody E-ECD E-ΔCT Ala1 Ala2 E 2968 6559 3454 14662 3388 29 2968G 2149375 9109 129 22 2969 2014 1857 7509 5671 3021 2969G 1347 610 6133 49422513 2970 5518 1324 14214 607 32 2970G 4683 599 12321 506 31 2971 501490 2506 2017 754 2971G 578 265 2457 2062 724 2972 2164 2158 8408 64093166 2972G 1730 992 6364 4602 2146 2973 3527 1148 3967 44 84 2973G 1294276 1603 28 44 2974 1766 722 8821 241 19 2974G 2036 228 8172 135 26 29751990 1476 8669 6134 2468 2975G 890 315 4194 3987 1376 2976 147 140 9961079 181 2976G 1365 460 6024 3929 1625

The anti-Antigen E common light chain antibody supernatants exhibitedhigh specific binding to the beads linked to Antigen E-ECD. For thesebeads, the negative control mock supernatant resulted in negligiblesignal (<10 MFI) when combined with the Antigen E-ECD bead sample,whereas the supernatants containing anti-Antigen E common light chainantibodies exhibited strong binding signal (average MFI of 2627 for 98antibody supernatants; MFI>500 for 91/98 antibody samples).

As a measure of the ability of the selected anti-Antigen E common lightchain antibodies to identify different epitopes on the ECD of Antigen E,the relative binding of the antibodies to the variants were determined.All four Antigen E variants were captured to the anti-myc LUMINEX™ beadsas described above for the native Antigen E-ECD binding studies, and therelative binding ratios (MFI_(variant)/MFI_(Antigen E-ECD)) weredetermined. For 98 tested common light chain antibody supernatants shownin Tables 12 and 13, the average ratios(MFI_(variant)/MFI_(Antigen E-ECD)) differed for each variant, likelyreflecting different capture amounts of proteins on the beads (averageratios of 0.61, 2.9, 2.0, and 1.0 for Antigen E-ΔCT, Antigen E-Ala1,Antigen E-Ala2, and Mf Antigen E, respectively). For each proteinvariant, the binding for a subset of the 98 tested common light chainantibodies showed greatly reduced binding, indicating sensitivity to themutation that characterized a given variant. For example, 19 of thecommon light chain antibody samples bound to the Mf Antigen E withMFI_(variant)/MFI_(Antigen E-ECD) of <8%. Since many in this groupinclude high or moderately high affinity antibodies (5 with K_(D) ⁻<5nM, 15 with K_(D)<50 nM), it is likely that the lower signal for thisgroup results from sensitivity to the sequence (epitope) differencesbetween native Antigen E-ECD and a given variant rather than from loweraffinities.

These data establish that the common light chain antibodies described inTables 5 and 6 represent a diverse group of Antigen-E-specific commonlight chain antibodies that specifically recognize more than one epitopeon Antigen E.

Example 11 Light Chain Shuffling in Common Light Chain Antibodies

Heavy chains of selected antigen-specific common light chain antibodieswere tested for binding to Antigen E after repairing the heavy chainswith either a germline Vκ1-39Jκ5 or a germline Vκ3-20Jκ1 engineeredlight chain (as described in Example 1).

Briefly, 247 heavy chains of Antigen E-specific common light chainantibodies (Vκ1-39Jκ5 and Vκ3-20Jκ1) were transfected with either agermline Vκ1-39 or a germline Vκ3-20 engineered light chain andrescreened for binding to Antigen E by a LUMINEX™ assay (as described inExample 7 and Example 10). Binding to Antigen E was confirmed byBIACORE™ (as described in Example 9). The results are shown in Table 15.

As shown in this Example, twenty-eight common light chain antibodiesspecific for Antigen E were capable of binding to Antigen E whenrepaired with a germline form of the light chain.

TABLE 15 Original Repaired Light Chain Light Chain No. Tested No.Confirmed Binders 1-39 1-39 198 23 3-20 3-20 49 5

Example 12 Heavy Chain Gene Usage and Somatic Hypermutation Frequency inCommon Light Chain Antibodies

Heavy and light chain sequences (>6000) of antibodies raised inVELCOIMMUNE® mice (e.g., U.S. Pat. No. 6,596,541 and U.S. Pat. No.7,105,348) were compiled with heavy and light chain sequences (>600) ofcommon light chain antibodies obtained by a multi-antigen immunizationscheme employing the engineered light chain mice (described above) tocompare heavy chain gene segment usage and somatic hypermutationfrequencies of the antibody chains.

Heavy Chain Gene Usage. Heavy and light chain sequences obtained fromVELOCIMMUNE® mice containing a replacement of the endogenous mouse heavychain locus with human V_(H), D_(H), and J_(H) gene segments and areplacement of the endogenous mouse κ light chain locus with either theengineered germline Vκ1-39Jκ5 human light chain region or the engineeredgermline Vκ3-20Jκ1 human light chain region (as described in Example 2)immunized with a human cell-surface receptor (Antigen E), a heterodimerof two human cell-surface glycoproteins (Antigen F), a human cytokinereceptor (Antigen G) and a human tumor differentiation antigen (AntigenH) were analyzed for heavy chain gene segment usage and V_(H) and J_(H)gene segments were recorded. Results are shown in Tables 16-18.Percentages in Tables 16-18 represent rounded values and in some casesmay not equal 100% when added together.

Table 16 sets forth the percent heavy chain family usage for antibodiesfrom VELCOIMMUNE® mice (VI), antibodies from VELCOIMMUNE® mice having acognate W1-39 light chain (VI-Vκ1-39), antibodies from Vκ1-39 engineeredlight chain mice (Vκ1-39), antibodies from VELCOIMMUNE® mice having acognate Vκ3-20 light chain (VI-Vκ3-20), and antibodies from Vκ3-20engineered light chain mice (Vκ3-20). Table 17 sets forth the percentV_(H) and J_(H) gene usage for antibodies from VELCOIMMUNE® mice (VI),antibodies from VELCOIMMUNE® mice having a cognate Vκ1-39 light chain(VI-Vκ1-39), antibodies from Vκ1-39 engineered light chain mice(Vκ1-39), antibodies from VELCOIMMUNE® mice having a cognate Vκ3-20light chain (VI-Vκ3-20), and antibodies from Vκ3-20 engineered lightchain mice (Vκ3-20). Table 18 sets forth the percent V_(H) gene usagefor antibodies from Vκ1-39 engineered light chain mice (Vκ1-39 Mice)from each immunization group (Antigens E, F, G and H) and the percentV_(H) gene usage for antibodies from Vκ3-20 engineered light chain mice(Vκ3-20 Mice) from selected immunization groups (Antigens E and G).

As shown in this Example, heavy chain gene usage for antigens tested inVκ1-39Jκ5 engineered light chain mice was characterized by apreponderance of V_(H) family III subgroups (V_(H)3-7, V_(H)3-9,V_(H)3-11, V_(H)3-13, V_(H)3-20, V_(H)3-23, V_(H)3-30, V_(H)3-33 andV_(H)3-48). Notable usage of other V_(H) family subgroups wascharacterized by usage of V_(H)1-18, V_(H)1-69, V_(H)2-5, V_(H)4-59 andV_(H)6-1. For antigens tested in Vκ3-20Jκ1 engineered light chain mice,heavy chain gene usage was characterized by a preponderance of V_(H)family III, V_(H) family IV and V_(H) family V subgroups (V_(H)3-11,V_(H)3-30, V_(H)3-33, V_(H)4-39, V_(H)4-59 and V_(H)5-51). Notable usageof other V_(H) family subgroups was characterized by usage of V_(H)1-18,V_(H)1-69, V_(H)2-70 and V_(H)6-1.

Somatic Hypermutation Frequency. Heavy and light chains from antibodiesraised in VELCOIMMUNE® mice and the engineered light chain mice(described above) were aligned to germline sequences according to theheavy and light chain gene usage demonstrated for each heavy and/orlight chain. Amino acid changes for each framework region (FW) andcomplementarity determining region (CDR) for both heavy and light chainof each sequence were calculated. Results are shown in Tables 19-22.Percentages in Tables 21-24 represent rounded values and in some casesmay not equal 100% when added together.

Table 19 sets forth the number of amino acid (AA) changes observed ineach FW and CDR region of heavy chains of antibodies from VELCOIMMUNE®mice, heavy chains of antibodies from Vκ1-39 engineered light chain mice(Vκ1-39 Mice) and heavy chains of antibodies from Vκ3-20 engineeredlight chain mice (Vκ3-20 Mice). Table 20 sets forth the number of aminoacid (AA) changes observed in each FW and CDR region of light chains ofantibodies from VELCOIMMUNE® mice, the light chain of antibodies fromVκ1-39 engineered mice (Vκ1-39 Mice) and the light chain of antibodiesfrom Vκ3-20 engineered mice (Vκ3-20 Mice). Table 21 sets forth thenumber of amino acid (AA) changes observed in each FW and CDR region ofheavy chains of antibodies from Vκ1-39 engineered light chain mice(Vκ1-39 Mice) for selected immunization groups (Antigens E, F and H).Table 22 sets forth the number of amino acid (AA) changes observed ineach FW and CDR region of heavy chains of antibodies from Vκ3-20engineered light chain mice (Vκ3-20 Mice) for selected immunizationgroups (Antigens E and G).

TABLE 16 V_(H) Family VI VI-Vκ1-39 Vκ1-39 VI-Vκ3-20 Vκ3-20 1 9.0 14.83.3 7.1 4.9 2 2.2 1.8 4.6 0 1.6 3 77.8 69.8 77.3 61.4 29.5 4 8.4 8.311.2 27.1 39.3 5 0.9 0 0.7 4.3 23.0 6 1.7 5.3 3.0 0 1.6

TABLE 17 VI VI-Vκ1-39 Vκ1-39 VI-Vκ3-20 Vκ3-20 V_(H) Gene 1-2 3.9 8.3 02.9 0 1-3 0 0 0 0 0 1-8 1.3 0.6 0 1.4 0 1-18 3.0 0.6 1.3 2.1 1.6 1-240.4 3.6 0 0.7 0 1-46 0.1 0 0 0 0 1-58 0 0 0 0 0 1-69 0.3 1.8 2.0 0 3.32-5 1.9 0 4.6 0 0 2-26 0.2 1.8 0.0 0 0 2-70 0.1 0 0 0 1.6 3-7 3.0 14.8 01.4 0 3-9 8.5 3.6 29.6 16.4 0 3-11 5.4 10.7 0 7.1 1.6 3-13 3.2 1.8 0.72.1 0 3-15 4.0 4.7 0.3 0.7 0 3-20 1.0 0.6 0.3 5.0 0 3-21 0.8 0.6 0 2.1 03-23 20.4 8.9 3.3 8.6 0 3-30 17.6 4.1 35.2 12.9 1.6 3-33 12.6 14.8 0 5.026.2 3-43 0.2 0.6 0 0 0 3-48 0.8 1.2 7.2 0 0 3-53 0.3 3.6 0.3 0 0 3-64 00 0.3 0 0 3-72 0 0 0 0 0 3-73 0 0 0 0 0 4-31 2.7 0 0.7 8.6 0 4-34 1.80.6 0.3 14.3 0 4-39 1.6 0.6 3.0 2.1 14.8 4-59 2.3 7.1 7.2 2.1 24.6 5-510.9 0 0.7 4.3 23.0 6-1 1.7 5.3 3.0 0 1.6 J_(H) Gene 1 1.5 1.2 7.1 0 0 24.5 2.4 0.7 5.0 26.9 3 10.5 16.6 13.1 13.6 26.9 4 44.0 34.3 32.3 50.79.6 5 9.6 10.1 16.8 7.9 1.9 6 29.7 35.5 30.0 22.9 34.6

TABLE 18 Vκ1-39 Mice Vκ3-20 Mice Antigen Antigen Antigen Antigen AntigenAntigen V_(H) Gene E F G H E G 1-2  0 0 0 0 0 0 1-3  0 0 0 0 0 0 1-8  00 0 0 0 0 1-18 0 0 0 8.3 0 3.1 1-24 0 0 0 0 0 0 1-46 0 0 0 0 0 0 1-58 00 0 0 0 0 1-69 2.9 0 25.0 0 0 6.3 2-5  8.2 0 0 0 0 0 2-26 0 0 0 0 0 02-70 0 0 0 0 0 3.1 3-7  0 0 0 0 0 0 3-9  1.2 98.8 0 14.6 0 0 3-11 0 0 00 0 3.1 3-13 0.6 0 25.0 0 0 0 3-15 0 1.2 0 0 0 0 3-20 0 0 25.0 0 0 03-21 0 0 0 0 0 0 3-23 4.1 0 25.0 4.2 0 0 3-30 62.9 0 0 0 3.4 0 3-33 0 00 0 13.8 37.5 3-43 0 0 0 0 0 0 3-48 0.6 0 0 43.8 0 0 3-53 1.6 0 0 0 0 03-64 1.6 0 0 0 0 0 3-72 0 0 0 0 0 0 3-73 0 0 0 0 0 0 4-31 0 0 0 4.2 0 04-34 0 0 0 2.1 0 0 4-39 5.3 0 0 0 31.0 0 4-59 11.8 0 0 4.2 3.4 43.8 5-511.2 0 0 0 48.3 0 6-1  0 0 0 18.8 0 3.1

TABLE 19 Heavy Chains of Antibodies from VELCOIMMUNE ® Mice # AA ChangesFW1 CDR1 FW2 CDR2 FW3 CDR3 0 63 32 36 26 12 82 1 23 32 41 31 22 17 2 925 17 23 27 1 3 4 10 5 16 13 0 4 0 1 1 3 12 0 >5 1 0 0 1 14 0 HeavyChains of Antibodies from Vκ1-39 Mice # AA Changes FW1 CDR1 FW2 CDR2 FW3CDR3 0 65 8 34 30 9 37 1 25 26 35 34 19 54 2 9 44 23 20 33 9 3 1 19 8 1222 0 4 0 3 0 5 11 0 >5 1 0 0 0 7 0 Heavy Chains of Antibodies fromVκ3-20 Mice #AA Changes FW1 CDR1 FW2 CDR2 FW3 CDR3 0 57 8 54 16 8 93 141 43 34 39 21 7 2 2 25 10 18 20 0 3 0 15 2 21 13 0 4 0 10 0 3 20 0 >5 00 0 2 18 0

TABLE 20 Light Chains of Antibodies from VELCOIMMUNE ® Mice # AA ChangesFW1 CDR1 FW2 CDR2 FW3 CDR3 0 65 24 49 60 33 23 1 24 20 34 31 27 38 2 927 16 9 18 28 3 1 20 1 0 14 7 4 0 7 0 0 4 3 >5 1 1 0 0 3 0 Light Chainsof Antibodies from Vκ1-39 Mice # AA Changes FW1 CDR1 FW2 CDR2 FW3 CDR3 091 75 80 90 71 63 1 9 19 17 10 21 27 2 0 5 1 1 5 8 3 0 0 1 0 2 1 4 0 0 00 2 1 >5 0 0 0 0 0 0 Light Chains of Antibodies from Vκ3-20 Mice #AAChanges FW1 CDR1 FW2 CDR2 FW3 CDR3 0 90 47 93 97 63 57 1 10 27 3 3 20 432 0 27 3 0 17 0 3 0 0 0 0 0 0 4 0 0 0 0 0 0 >5 0 0 0 0 0 0

TABLE 21 Heavy Chains of Anti-Antigen E Antibodies from Vκ1-39 Mice # AAChanges FW1 CDR1 FW2 CDR2 FW3 CDR3 0 75 8 49 41 14 36 1 21 25 33 35 2552 2 4 43 14 18 28 12 3 0 20 4 5 16 0 4 0 5 0 1 12 0 >5 1 0 0 0 5 0Heavy Chains of Anti-Antigen F Antibodies from Vκ1-39 Mice # AA ChangesFW1 CDR1 FW2 CDR2 FW3 CDR3 0 52 0 6 6 2 15 1 35 24 32 35 15 78 2 11 5946 22 49 7 3 0 17 16 24 29 0 4 0 0 0 12 4 0 >5 1 0 0 0 1 0 Heavy Chainsof Anti-Antigen H Antibodies from Vκ1-39 Mice # AA Changes FW1 CDR1 FW2CDR2 FW3 CDR3 0 54 21 29 33 4 77 1 17 35 50 27 6 23 2 23 21 15 21 25 0 36 21 4 15 27 0 4 0 2 2 2 15 0 >5 0 0 0 2 23 0

TABLE 22 Heavy Chains of Anti-Antigen E Antibodies from Vκ3-20 Mice # AAChanges FW1 CDR1 FW2 CDR2 FW3 CDR3 0 79 17 62 24 17 90 1 21 28 34 55 3110 2 0 28 3 21 24 0 3 0 14 0 0 10 0 4 0 14 0 0 3 0 >5 0 0 0 0 14 0 HeavyChains of Anti-Antigen G Antibodies from Vκ3-20 Mice # AA Changes FW1CDR1 FW2 CDR2 FW3 CDR3 0 38 0 47 9 0 97 1 59 56 34 25 13 3 2 3 22 16 1616 0 3 0 16 3 41 16 0 4 0 6 0 6 34 0 >5 0 0 0 3 22 0

1. A mouse that expresses a population of antigen-specific antibodies inresponse to challenge with an antigen, wherein all of the immunoglobulinlight chains of the population of antigen-specific antibodies comprise ahuman light chain variable (V_(L)) region derived from the same singlehuman V_(L) gene segment and the immunoglobulin heavy chains comprise ahuman heavy chain variable (V_(H)) region derived from a human V_(H)gene segment selected from V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18,V_(H)1-24, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5, V_(H)2-26,V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13, V_(H)3-15,V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)3-33, V_(H)3-43,V_(H)3-48, V_(H)3-53, V_(H)3-64, V_(H)3-72, V_(H)3-73, V_(H)4-31,V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)5-51, V_(H)6-1.
 2. The mouse ofclaim 1, wherein the same single human V_(L) gene segment is a humanVκ1-39 gene segment.
 3. The mouse of claim 1, wherein the same singlehuman V_(L) gene segment is a human Vκ3-20 gene segment.
 4. The mouse ofclaim 1, wherein all of the immunoglobulin light chains comprise a humanlight chain J (J_(L)) gene segment.
 5. The mouse of claim 4, wherein thehuman J_(L) gene segment is selected from a human Jκ1 and a Jκ5 genesegment.
 6. The mouse of claim 1, wherein the mouse lacks a sequenceselected from a mouse immunoglobulin V_(L) gene segment, a mouseimmunoglobulin J_(L) gene segment, and a combination thereof.
 7. Themouse of claim 1, wherein the human V_(L) region is operably linked to ahuman, mouse, or rat immunoglobulin light chain constant (C_(L)) region.8. The mouse of claim 1, wherein the human V_(L) region is expressedfrom an endogenous immunoglobulin light chain locus.
 9. The mouse ofclaim 1, wherein the human V_(H) region is operably linked to a human,mouse, or rat immunoglobulin heavy chain constant (C_(H)) region. 10.The mouse of claim 1, wherein the human V_(H) region is expressed froman endogenous immunoglobulin heavy chain locus.
 11. A mouse thatexpresses a population of antigen-specific antibodies in response tochallenge with an antigen, wherein all of the immunoglobulin lightchains of the population of antigen-specific antibodies comprise a humanV_(L) region derived from a human Vκ1-39 gene segment and theimmunoglobulin heavy chains comprise a human heavy chain variable(V_(H)) region derived from a human V_(H) gene segment selected fromV_(H)1-18, V_(H)1-69, V_(H)2-5, V_(H)3-9, V_(H)3-13, V_(H)3-15,V_(H)3-20, V_(H)3-23, V_(H)3-30, V_(H)3-48, V_(H)3-53, V_(H)3-64,V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)5-51 and V_(H)6-1. 12.The mouse of claim 11, wherein the human V_(L) region comprises a Jκ5sequence.
 13. The mouse of claim 11, wherein the mouse lacks a mouseimmunoglobulin V_(L) and/or J_(L). gene segment.
 14. The mouse of claim11, wherein the human V_(L) region is operably linked to a human, rat,or mouse immunoglobulin light chain constant (C_(L)) region.
 15. Themouse of claim 11, wherein the human V_(L) region is expressed from anendogenous immunoglobulin light chain locus.
 16. The mouse of claim 11,wherein the human V_(H) region comprises a sequence that encodes a humanJ_(H) region selected from a human J_(H)1, J_(H)2, J_(H)3, J_(H)4,J_(H)5, and J_(H)6 gene segment.
 17. The mouse of claim 11, wherein thehuman V_(H) region is operably linked to a human, rat, or mouseimmunoglobulin heavy chain constant (C_(H)) region.
 18. The mouse ofclaim 11, wherein the human V_(H) region is expressed from an endogenousimmunoglobulin heavy chain locus.
 19. A mouse that expresses apopulation of antigen-specific antibodies in response to challenge withan antigen, wherein all of the immunoglobulin light chains of thepopulation of antigen-specific antibodies comprise a human V_(L) regionderived from a human Vκ3-20 gene segment and the immunoglobulin heavychains comprise a human heavy chain variable (V_(H)) region derived froma human V_(H) gene segment selected from V_(H)1-18, V_(H)1-69,V_(H)2-70, V_(H)3-11, V_(H)3-30, V_(H)3-33, V_(H)3-53, V_(H)4-39,V_(H)4-59, V_(H)5-51 and V_(H)6-1.
 20. The mouse of claim 19, whereinthe human V_(L) region comprises a Jκ5 sequence.
 21. The mouse of claim19, wherein the mouse lacks a mouse immunoglobulin V_(L) and/or J_(L)gene segment.
 22. The mouse of claim 19, wherein the human V_(L) regionis operably linked to a human, rat, or mouse immunoglobulin light chainconstant (C_(L)) region.
 23. The mouse of claim 19, wherein the humanV_(L) region is expressed from an endogenous immunoglobulin light chainlocus.
 24. The mouse of claim 19, wherein the human V_(H) regioncomprises a sequence that encodes a human J_(H) region selected from ahuman J_(H)2, J_(H)3, J_(H)4, J_(H)5, and J_(H)6 gene segment.
 25. Themouse of claim 19, wherein the human V_(H) region is operably linked toa human, rat, or mouse immunoglobulin heavy chain constant (C_(H))region.
 26. The mouse of claim 19, wherein the human V_(H) region isexpressed from an endogenous immunoglobulin heavy chain locus.